PS01

Probing the DNA kink structure induced by the

hyperthermophilic chromosomal protein Sac7d using site-directed

mutagenesis and X-ray crystallography

 

Chin-Yu Chen, 1,2 Ting-Wan Lin,1 Chia-Cheng Chou,1 Tzu-Ping Ko1 and Andrew H.-J. Wang 1,3

1Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan and 2 Dept. Chemistry, National Taiwan University, Taiwan and 3Institute of Biochemical Sciences, National Taiwan University

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

The protein Sac7d belongs to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Previous studies show that Sac7d causes a single-step sharp kink in DNA (~60o) via the intercalation of both Val26 and Met29. In this paper, site-directed mutagenesis techniques were applied to change the side chains of Val26 and Met29 systematically to either smaller or larger sizes. The crystal structures of Sac7d mutants (V26A, M29A, V26A/M29A, M29F and V26F/M29F) and GCGATCGC complexes have been determined and refined at 2.25 Å, 2.2 Å, 1.45 Å, 1.9 Å and 1.5 Å, respectively. The crystal structures of Sac7d single mutants (V26A, M29A, and M29F) and the other DNA GTAATTAC complexes also have been determined and well refined at 2.2 Å, 1.6 Å and 1.7 Å, respectively. DNA bending has long been recognized as an important component of biological activity. The Sac7d mutants display two kinking modes of DNA conformations that are “smooth bending” and “kinked bending” into minor groove of DNA duplex. The DNA binding patterns and unit cells of the V26A, M29A single mutants are similar to those of wild type. Without stacking with one of the base pairs, the Phe29 side chain of M29F mutant penetrates into the C2pG3 (A3pA4) step vertically, together with the Vla 26, induces the largest single-step sharp kink (~70o) and disrupts the stacking of two adjacent base pairs. For the double mutants, the unit cells of crystals are both smaller than that of wild type and the crystal structures reveal some surprising unprecedented change. Instead of binding at the C2pG3 step as observed in the wild type Sac7d, the V26A/M29A protein binds at the G3pA4 step and has smaller kink angle (~50o). The hydrophobic side chain of the residue Phe26 in V26F/M29F-GCGATCGC complex intercalates deeply into the gap of DNA bases by p-p stacking interactions with G3 base, whereas the side chain of Phe29 can not insert itself into C2pG3 most likely due of the steric hindrance effect. The Phe29 is packed against the G15 ribose with van der Waals contact. Thermodynamic studies show that all mutants still bind to DNA, but with weaker affinity.

 

 

PS02

The coil-to-structural transition of the prion peptide can be affected by a single O-linked sugar

 

Rita P.-Y. Chen1*, Chun-Cheng Lin2, Yin-Ting Chang2, Su-Ching Lin2 and Sunney I. Chan2

1Institute of Biological Chemistry and 2Institute of Chemistry, Academia Sinica, Taipei, Taiwan, R. O. C.

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

It has been known that the structural transition from PrPC to PrPSc leads to the prion formation.  This putative conformational change challenges the central dogma of the protein folding theory - ‘one sequence, one structure’.  Generally, scientists believe that there must be either a post-translational modification or environmental factors involved in this event.  However, all the efforts to solve the mystery of the PrPC to PrPSc transition have ended in vain so far.  Here we provide evidence linking O-linked glycosylation to the structural transition based on prion peptide studies.  We find that the O-linked a-GalNAc at Ser-135 suppresses the formation of amyloid fibril formation of the prion peptide at physiological salt concentrations, whereas the peptide with the same sugar at Ser-132 shows the opposite effect.  Moreover, this effect is sugar specific.  Replacing a-GalNAc with b-GlcNAc does not yield the same effect.

 

PS03

Catalytic Mechanism Revealed by the Crystal Structure of Undecaprenyl

Pyrophosphate Synthase in Complex with Sulfate, Magnesium and Triton

 

Tzu-Ping Ko*, Sing-Yang Chang, Po-Huang Liang, and Andrew H.-J. Wang

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

128 Academia Road Section 2, Nankang, Taipei 11529, Taiwan

 

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate (UPP) via condensation with eight isopentenyl pyrophosphates (IPP). UPP is a lipid carrier to mediate bacterial peptidoglycan synthesis. UPPs from Escherichia coli is a dimer and each subunit consists of 253 amino-acid residues. The chain length of the product is modulated by a hydrophobic active-site tunnel surrounded by two a-helices and four b-strands. New crystals of E. coli UPPs were grown in the presence of ammonium sulfate, magnesium chloride and Triton X-100, but turned out to be isomorphous to the previously solved apo-enzyme crystals. The crystals belong to the orthorhombic space group of P212121 with unit-cell dimension of a = 63.96 Å, b = 67.42 Å, c = 111.76 Å. The structure was refined to 1.73 Å resolution with R and Rfree values of 0.176 and 0.208, respectively. It showed bound sulfate and magnesium ions, as well as Triton X-100 molecules. The amino acid residues 72 – 82, which encompass an essential catalytic loop not seen in the previously determined structure, also became visible in one subunit. The sulfate ions suggest locations of the pyrophosphate groups of FPP and IPP in the active site. The site for binding FPP corresponds to a highly conserved structural P-loop in the enzyme. The Mg2+ is chelated by His199 and Glu213 from different subunits and possibly plays a structural rather than catalytic role. However, the metal ion is near the IPP-binding site and double mutation of His199 and Glu213 to alanines showed a remarkable increase of Km value for IPP. Inside the tunnel, one Triton surrounds the top portion of the tunnel and the other occupies the bottom part. These two Triton molecules may mimic the hydrocarbon moiety of the UPP product in the active site, with the cis-isoprenyl groups on the top and the all-trans farnesyl part in the bottom. Based on these observations, a possible reaction mechanism for UPPs catalysis is proposed, in which the side chains of Asp26 and His43 play the key roles of general base and general acid, respectively, to deprotonate and protonate the substrate molecules.

 

 

PS04

Structure detemination and DNA-binding properties of Sso7C4, a thermostable protein from the archaeon Sulfolobus solfataricus

 

Chun-Hua Hsu1, Jeu-Ming P. Yuann1, Andrew H. Wang1*

1Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

Sso7c4, a protein of the hyperthermophilic organism Sulfolobus solfataricus, has been proposed to play a regulatory role in gene transcription in Archaea.  However, very little is known regarding the transcription process in Archaea.  And Sso7c4, along with other proteins isolated so far, appears to be the first repressor-like proteins described in this kingdom. One possible way to study the properties of Sso7c4 and how it is involved in the transcription of Sulfolobus solfataricus is to determine its 3-dimensional structure. The protein is extremely stable to heat, acid and chemical agents. We have determined the solution structure of Sso7c4 using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy.  The protein consists of a triple-stranded anti-parallel beta-sheet and a helix. In addition, Sso7c4 was successfully crystallized and diffracted by x-ray radiation to high resolution (1.90 Å).  The existing x-ray crystallographic evidence shows that the space group of Sso7c4 crystal was p212121 with a, b, c, and α, β, γ being 54.2, 55.6, 92.7, and 90, 90, 90, respectively. Other biophysical methods (CD, SPR, etc.) have been carried out to characterize its DNA binding properties.

 

  

PS05

Structure and Backbone Dynamics of proteins from the transacylase component of Human Mitochondrial Branched-Chain

alpha-Ketoacid Dehydrogenase

 

Chi-Fon Chang, Shin-Jye Lee, Yi-Jan Lin, Bin-Nan Chen, Tai-Huang Huang

1Institute of Biomedical Sciences, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan

and 2National Taiwan University, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

  Di-domain of the transacylase component (E2) of human mitochondrial branched-chain a-ketoacid dehydrogenase complex (BCKD complex) contains the sites of known human mutations that cause inherited maple syrup disease (MSUD). The E2 component of BCKD complex consists of three independently folded domains, i.e. the N-terminal lipoyl-bearing domain (hbLBD), the interim E1/E3-binding domain, and the C-terminal inner core domain. These three domains are linked through flexible linkers permitting hbLBD to play a central role in substrate channeling by visiting three sites in cognate E1, E2 and E3 components. We have solved the three-dimensional solution structure of hbLBD and hbCBD by multidimensional nuclear magnetic resonance spectroscopy. The solution structure of hbLBD is a flattened beta-barrel formed by two four-stranded anti-parallel beta sheets. The lipoylated lysine residue is prominently situated on a tight turn at the corner of one of the beta-sheets for interactions with E1. hbCBD is a remarkably small domain of 49 amino acids and its structure was found to made up of two helices connected by a helix-like turn and an irregular loop. To assess the independence of the hbLBD and hbCBD and to determine the flexibility of the linker region we have measured the backbone 15N-T1, T2, and 15N-1H NOE for a di-domain consisting of hbLBD, hbCBD and the linker region. These data were analyzed using reduced spectral density mapping formalism to extract the backbone dynamics information. We have also investigated the structures and folding behaviors of a series of hbLBD mutatants that cause MSUD. The results of which will be discussed as well.

 

 

PS06

Identification of A Dimer-Forming Region In SARS-CoV Nucleocapsid Protein

 

Chung-ke Chang1*, Tsan-hung Yu2, Hsin-hao Hsiao1, Hsiao-yi Hung2, Shin-Jye Lee1, Shih-Che Sue1 and Tai-huang Huang1.

1Institute of Biomedical Sciences, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan

and 2National Taiwan University, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

The SARS coronavirus (SARS-CoV) nucleocapsid (N) protein is an RNA-binding protein and a major structural component of SARS-CoV.  The PONDR program predicts a well-ordered region at the C-terminus of SARS-CoV N protein.  Preliminary NMR spectroscopy data of a clone containing this region shows that it is indeed a structured region.  Based on these findings, we cloned a number of N protein fragments centered around this region.  A combination of NMR T2 measurements, analytical gel filtration chromatography and cross-linking experiments were undertaken to characterize these fragments.  T2 measurements yielded a time constant of 30-50 ms, whereas chromatography results imply existence of oligomeric species under our experimental conditions.  Cross-linking experiments showed formation of dimers and possibly higher order oligomers.  Taken together, our results indicate that the C-terminal well-ordered region of SARS-CoV N protein is able to form dimers and might be implicated in SARS-CoV N protein oligomerization.

 

 

PS07

Structural and Functional Characterization of Human Hepatoma-Derived Growth Factor (hHDGF)

 

Shih-Che Sue*, Tzu-Feng Fu, Jeou-Yuan Chen, and Tai-huang Huang,

Institute of Biomedical Sciences, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan

 

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a new protein family with growth-stimulating activity toward various cell lines such as fibroblasts, endothelial cells and smooth muscle cells. hHDGF, the first HRP discovered, was initially identified from the conditioned medium of human hepatoma-derived Huh7 cells. By representational analysis, we found that the expression of hHDGF is up-regulated in human gastric tumor, suggesting a possible role of hHDGF in oncogenesis. Efforts are made to elucidate the structural and functional mechanisms of hHDGF. Based on SPR experiment and heparin affinity chromatography, we identified the N-terminal domain (N terminus to Gly100) as a novel heparin/heparan sulphate-binding domain with a Kd value ~ 20 nM, whereas the fragment of the C-terminal domain (Gln101 to C terminus) had no heparin-binding ability. NMR 15N-HSQC spectra showed that the heparin-binding domain had a well-defined structure, but C-terminal region is disordered. However, the disordered C-terminal region exhibited transformation activity to enhance cell saturation density and growth rate in H1299 cells, while N-terminal domain showed no effect. Since all HRPs share a 60% - 80% sequence identity at the N-terminal domain, we propose that the conserved N-terminal domains of all HRPs may play a similar role as an interaction partner with heparin-like molecule on the cell surface. The non-conserved C-terminal parts may confer various biological functions and/or allow interaction with multiple cellular targets. Finally, we report the first solution NMR structure of the conserved N-terminal domain. In conclusion, our results have provided substantial insight into the molecular mechanism of hHDGF in relation to oncogenesis and have also raised several intriguing questions regarding the functions of the various domains of HRPs.

 

 

PS08

Backbone Dynamics and Thermodynamics of Escherichia coli Theosterase / Protease I – A temperature-dependent 15N-NMR study

 

C.Y. Chou2, S.I. Tyukhtenko1, Y.T. Huang1 and T. H. Huang1,2

1Institute of Biomedical Sciences, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan and

2National Taiwan Normal University, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

E. coli thioesterase/protease I (TEP-I) is a serine protease of the SGNH-hydrolase family. We have applied 15N nuclear magnetic spin relaxation method to investigate the multiple time scale temperature dependent backbone motions in TEP-I. Laboratory frame relaxation data at 278K, 285K, 295K, and 310K were analyzed using modelfree and reduced spectral density approaches. Analysis of the relaxation measurements yields order parameters (S2) that reflect the degree of spatial restriction for backbone amide H-N vectors. The entropy characterizing the motional behavior of a specific N-H vector of our target protein can be further calculated from temperature dependence S2. We found that the TEP-I molecule as a whole is rather rigid. On the other hand, the active site pocket exhibits high degree of flexibilities with high entropy. The thermodynamic parameters extracted from the analysis will be presented. The biological consequences and the changes in dynamics and thermodynamics at the intermediate stages of catalysis, ie. the Michaelis complex and tetrahedral complex, will be discussed.

 

 

PS09

Folding Mechanism and Thermodynamics of the Lypoyl-Bearing Domain

of the E2 Component of Human Mitochondrial Branched

Chain a-Ketoacid Dehydrogenase

Shin-Jye Lee1, Mandar T. Naik1, Yu-Chu Chang1, Bin-Nan Chen2 and Tai-huang Huang1

1Institute of Biomedical Sciences, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan

and 2National Taiwan University, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

The Lipoic Acid Bearing Domain (hb LBD) of the E2 component of human mitochondrial branched chain α-ketoacid dehydrogenase (BCKD) complex is known to play a central role in substrate channeling in the metabolism of α-ketoacid. We have employed CD and fluorescence techniques to study the folding mechanism of hbLBD. Both studied suggested mono exponential kinetics without any burst phase. Moreover the thermodynamic parameters ΔGH20 and m value obtained from the kinetic analysis are consistent with the equilibrium measurements, thus ruled out populated kinetic or equilibrium intermediates on folding pathway of thisβbarrel domain under experimental conditions. We concluded that the hb LBD folding follows a two-step mechanism without detectable presence of stable or kinetic intermediates. We have also employed thermal denaturation in the presence of urea and isothermal urea denaturation titrations to study the conformational stability underlying the unfolding of this smallβbarrel domain and have evaluated various thermodynamic parameters defining the equilibrium unfolding. The linear extrapolation model successfully describes the two-step; native state « denatured state unfolding transition of hbLBD. The average temperature of maximum stability of hbLBD is estimated as 295.6 ± 0.9 K. Cold denaturation of hbLBD is also predicted and discussed. Some more recent studies on conformational stabilities of hbLBD mutants with pathological consequences will be presented also.

 

 

PS10

NMR Investigation of the Structure and Catalytic Mechanism of a Serine     Protease-E.coli Thioesterase/Protease I

 

Sergiy I. Tyukhtenko1*, Alexandra V. Litvinchuk1, Chi-Fon Chang1, Jei-Fu Shaw2 and Tai-huang Huang1

1Inst. Biomedical Sciences and 2Inst. Botany, Academia Sinica, Nan-Kang, Taipei, 11529, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

E.coli thioesterase/protease I (TEP-I) belongs to a new subclass of lipolytic enzymes of the serine hydrolase superfamily. Ser10, Asp154 and His157 have been identified as its catalytic triad residues. We have employed NMR techniques to characterize the structure, dynamics and catalytic mechanism of TEP-I. We showed that mechanism-based inhibitor diethyl p-nitrophenyl phosphate (DENP) interacts with TEP-I in two steps, the fast formation of the Michaelis complex followed by a slow conversion to the transition state analogue complex, permitting detailed structural study of both complexes. Perturbation induced by DENP binding was found to confine primarily to the active site region. 31P NMR data revealed significant conformational change for inhibitor in the Michaelis complex accompanying the conformational perturbations around TEP-I active site. We have also detected several low-field exchangeable 1H resonances in TEP-I. The functional significance of the combined study will be discussed.

 

 

  

PS11

DNA Binding and Degradation by the HNH Protein ColE7

 

Kuo-Chiang Hsia1, Kin-Fu Chak2, Po-Huang Liang3, Yi-Sheng Cheng1, Wen-Yen Ku1, and Hanna S. Yuan1

1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.

2Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan, ROC.

3Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, ROC.

 

The bacterial toxins, colicins, are antibacterial proteins released by E. coli when bacteria suffer from environmental stresses.  Colicin E7 (ColE7) belongs to the DNase type which kills bacteria by degrading target cells’ chromosomes and its DNase activity comes from C-terminal nuclease domain in which an HNH motif is located.  The HNH motif in ColE7 has been identified in hundreds of prokaryotic and eukaryotic endonucleases, involved in DNA homing, restriction, repair or chromosome degradation.  Here we report the crystal structure of the nuclease domain of ColE7 in complex with a duplex DNA at a resolution of 2.5 Å.  The crystal structure shows that the HNH motif is bound at the minor groove primarily to DNA phosphate groups at and beyond the 3’-side of the scissile phosphate.  This result provides a structural basis for sugar and sequence independent DNA recognition and the inhibition mechanism by inhibitor Im7, which blocks the substrate binding site but not the active site.  Based on biochemical results, ColE7 digests ssDNA with a length more than 7-bases, different from other non-specific endonucleases which usually digest DNA to monomers or dimers.  Structural comparison further shows that two families of endonucleases bind and bend DNA in a similar way to that of the HNH ColE7, indicating that endonucleases containing a “bba-metal” fold of active site possess a universal mode for protein-DNA interactions.

 

 

 

PS12

Structural Basis of the Lipid Binding in Rice Nonspecific Lipid Transfer Protein-1 Complexes from Oryza sativa

 

Hui-Chun Cheng*, Pei-Tsung Cheng, Peiyu Peng, Ping-Chiang Lyu and Yuh-Ju Sun*

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan, ROC

 

Nonspecific lipid transfer proteins (nsLTPs) facilitate the transfer of phospholipids, glycolipids, fatty acids, and steroids between membranes, with the binding affinity of a broad range.  Some nsLTPs exhibit anti-microbial activity in vitro.  Herein we report three crystal structures of rice nsLTP1 from Oryza sativa, complexed with myristic acid, palmitic acid and stearic acid.  This is the first rice nsLTP1 complex crystal structure has been reported.  One fatty acid binding site was observed in myristate and stearate complexes and two fatty acids binding sites were observed in palmitate complex.  The overall folding of rice nsLTP1 complexes belongs to the four-helix bundle with a long C-terminal segment, which is similar to other plant nsLTP1s.  The C-terminal loop region consists of the elasticity to accommodate various fatty acids and the number of molecules.  Meanwhile, the second lipid-binding site of rice nsLTP1 is depended on the amount of lipid molecules participating in the complex formation.  The most significant binding forces between the fatty acids and rice nsLTP1s proteins are the hydrophobic interactions from the binding cavity of proteins.  A significant conformational change of the C-terminal loop was found between the unliganded and liganded rice nsLTP1s. 

 

 

PS13

Preliminary X-ray Diffraction Analysis of Hydantoinase from Pig Liver and Agrobacterium radiobacter

 

Jia-Yin Tsai1*, Sheng-Kuo Chiang1, Cheng-Yang Huang2, Yuh-Shyong Yang2, Yuh-Ju Sun1

1Institute of Bioinformatics and Structural Biology & Department of Life Science,

National Tsing Hua University, Hsinchu, Taiwan 300, ROC

2Department of Biological Science and Technology, College of Science, National Chiao Tung University,

Hsinchu, Taiwan 300, ROC

 

Mammalian hydantoinase is the enzyme involved in uracil and thymine catabolism. A thermophilic hydantoinase from pig was purified and crystallized. One belongs to the triclinic P1 space group, with unit-cell parameters a = 96.35, b = 96.87, c = 154.87 Å, a= 82.10, b= 72.54, g= 77.19, and the other belongs to the orthorhombic C2221 space group, with unit-cell parameters a = 113.92, b = 157.22, c = 156.21Å. Bacterial hydantoinase is a zinc enzyme and used in industry as a biocatalyst for the production of D- or L-amino acids. A D-hydantoinase from Agrobacterium radiobacter was expressed in Escherichia coli, purified to homogeneity and crystallized. D-Hydantoinase was crystallized using ammonium sulfate as a precipitant. The crystal form belongs to tetragonal I4 space group, with unit-cell parameters a = b = 129.39Å, c = 173.31Å and diffracted to 2.6Å containing three molecule per asymmetric unit. Taking advantage of the zinc-binding protein, MAD data sets of zinc anomalous scattering were collected. In the future, combined homologous structure models and MAD phasing will be applied in the structure determination of D-Hydantoinase.

 

 

 

PS14

Conformation and Orientation of Somatostatin on Sodium Dodecyl

Sulfate micelle

 

Wei Jyun Chien

Department of Applied Chemistry, Chaoyang University of Technology, Wufeng, Taichung, Republic of China

No.168 Gefeng East Road, Wufeng, ,Taichung, Taiwan Republic of China

 

Conformation of a disulfide-linked 14-residue cyclic peptide, somatostatin, under micellar environment is simulated based on the constraints derived from NMR experiments. The central residues in bioactive region Phe7-Trp8-Lys9-Thr10 adopts either loose-defined type II’ b-turn or more conserved type I’ b-turn in the presence of SDS micelle. Upon binding of somatostatin to SDS micelles, Phe7-Trp8-Lys9-Thr10 segment shows the most obvious decrease in the base-catalyzed amide-proton exchange rates. The sequence-specific effects of micelle interaction are also observed in the homo-nuclear nonselective spin-lattice relaxation times. These results combined with paramagnetic broadening observation on peptide protons in the presence of spin-labeled lipids yield a detailed model of the interaction of somatostatin with lipid surface. The Asn5-Phe 11 fragment interacts with SDS micelle while the Phe6-Phe7-Trp8-Lys9 is close to the center of the micelle The association constant of somatostatin to SDS micelle, determined via pulsed-field-gradient NMR techniques, is about 1.1×103 M-1.

 

 

PS15

Conformation of Sodefrin: a Female-Attracting Peptide Pheromone in newt

 

Wei Jyun Chien, Chien-PinHsu

Department of Applied Chemistry, Chaoyang University of Technology, Wufeng, Taichung, Republic of China

No.168 Gefeng East Road, Wufeng, ,Taichung, Taiwan Republic of China

 

The conformation of the female-attracting pheromones, sodefrin first obtained from the abdominal gland of Cynops pyrrhogaster is investigated by CD and NMR spectroscopy. In aqueous solution, this decapeptide exists mainly as a random coil. The introduction of TFE resulted in an increase of the helix content. A primary structure derived from NMR experiments and distance geometry revealed the existence of a nascent helix in C-terminal of the peptide in 50% TFE solution. This result is consistent with the chemical shifts and temperature coefficients of backbone amide protons. Substitution of proline for alanine at position 3 caused a reduction in the helix content in 50% TFE. The deviation of the mutant structure from that of wild peptide is monitored by circular dichroism experiments and identified by a three dimensional curve, which was composed of the information from both variable temperature and TFE titration experiments. Based on these observations proline, at N-terminal of sodefrin, seems to be able to stabilize the helical structure in C-terminal of the short peptide.

 

 

PS16

DNA binding and cleavage by the periplasmic endonuclease Vvn from Vibrio vulnificus: A novel structure with a known active site

 

Chia-Lung Li1,2, Lien-I Hor3, Zee-Fen Chang2, Li-Chu Tsai1, Wei-Zen Yang1 and Hanna S. Yuan1,2

1Institute of Molecular Biology, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan and 2Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taiwan, 3Department of Microbiology and Immunology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan.

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

The Vibrio vulnificus nuclease, Vvn, is a non-specific periplasmic nuclease capable of digesting DNA and RNA. Vvn protects cells by preventing the uptake of foreign DNA during transformation. The crystal structure of the magnesium ion-bound Vvn and that of Vvn mutant H80A in complex with a duplex DNA and a calcium ion were resolved both at 2.3 Å resolution. Vvn has a novel mixed a/b topology containing four disulfide bridges. The overall structure of Vvn shows no similarity to other endonucleases, however, a known endonuclease motif containing a “bba-metal” fold is identified in the central cleft region. The crystal structure of the mutant Vvn/DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by about 20o. Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting at structural basis for its sequence-independent recognition of DNA and RNA. Based on the enzyme/substrate and enzyme/product structures observed in the mutant Vvn/DNA crystals, a catalytic mechanism is proposed in which the His80 functions as a general base that activates a water molecule to attack the scissile phosphate, with a magnesium ion involved in stabilization of the phosphoanion transition state and in protonation of the 3’ oxygen. This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and indicates how non-specific DNA-binding proteins may recognize DNA.

 

 

PS17

Crystal structure of CTX A3 and hexasaccharide heparin complex from

Naja atra

 

 

Hong-Hsiang Guan1,2* Shao-Chen Lee 2 Wen-Guey Wu 2 , Chung-Jung Chen 1*

1Synchrotron Radiation Research Center, Hsinchu, Taiwan

2 Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu, Taiwan

 

Cardiotoxin (CTX) is a major component of cobra toxin. Cobra cardiotoxins (CTXs) are basic proteins, composed of 60-62 amino acids, in which b-sheets form three finger-loop structures . When cobra bite animals, CTX can induce tissue inflammation. However, the CTX major target on cell membrane remains unclear. The previous studies have proven that the heparan sulfate is the most potential target for CTX on cell membranes. We have determined the complex structure of CTX A3 and hexasaccharide which shows the loop 2 of CTX A3 is the major heparin binding location. Positively-charged amino acid residues near the tip of loop 2 play an important role on binding heparin through ionic interactions with O-sulfates. It is suggested that the loop 2 is a heparin-binding loop. CTXs are aggregated on cell membrane through loop 2 binding to heparan sulfates to enhance its toxin. 

 

 

PS18

Purification and Crystallization of Natrin, a Cystein-rich Secretory Protein, from Naja atra

 

Yu-Lin Wang1,2*, Shao-Chen Lee 2 , King-Xiang Goh 2 , Wen-guey Wu 2 and Chun-Jung Chen 1*

1 Structural Biology Group, National Synchrotron Radiation Research Center, Hsinchu, Taiwan

2 Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu, Taiwan

No.101 Hsin-Ann Rd., Science-Based Industrial Park, Hsinchu 30077, Taiwan, R.O.C.

 

Cysteine-rich secretory proteins (CRISPs) may play a role in the innate immune system and are transcriptionally regulated by androgens in several tissues. They are mostly found in epididymis and granules of mammals . A number of snake venoms, from habu snake, erabu sea snake, Conus textile, etc., contain CRISP family proteins . Natrin has a cysteine-rich C-terminal tail and belongs to a family of CRISPs. We have purified the natrin protein from Naja atra (Taiwan cobra) venom using a three-step chromatography procedure. The protein is composed of 239 amino acid residues, which has an apparent molecular mass of 24941 Da with an alkaline pI value of 8.5 and consists of one single polypeptide chain as estimated by mass spectrometry and SDS-PAGE. The biological function of natrin is unclear so far. We have obtained natrin crystals and tend to determine the structural by X-ray crystallography to understand its special biological function in venom.  The purification and preliminary X-ray analysis will be discussed.

 

 

PS19

The 3-D Structure and Surface Properties of Human Post-Translational

Modifier Proteins SUMO-1/2/3

 

Wen-Chen Huang1*, Tzu-Ping Ko2, Steven S.-L. Li1 and Andrew H.-J. Wang2

1Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan

2Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei 11529 , Taiwan

1No.70 Lien-hai Rd. Kaohsiung 804, Taiwan

2No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

The SUMO protein was named Small Ubiquitin-like Modifier because its 3-D structure was similar to ubiquitin. In human, there are three isoforms of SUMO, namely, SUMO-1/2/3. The recombinant Δ1-8,93-95 SUMO-2 protein with 10 histidine residues at its N-terminus was expressed using E. coli. BL-21(DE3), purified at 4 °C and crystallized at room temperature. The surface properties of human SUMO-1/2/3 proteins and 3-D of structure were analyzed using computer modeling and X-ray diffraction technology.

The two-step purification by immobilized metal ion affinity chromatography (IMAC) was developed to yield Δ1-8,93-95 SUMO-2 protein that reached 60 mg/ml for crystallization. On protein expression, 120 mg protein was obtained from 6 L bacterial growth broth. Crystallization was achieved by the hanging-drop vapor diffusion method and obtained a single triangular plate polyhedron. The crystal (0.35x0.15x0.1 mm3) diffracted X-rays to 1.2 Å resolution. Analysis of the diffraction pattern suggests the crystal belongs to R3 space group and has unit cell parameters a= b=74.96 Å, c=33.23 Å, α=90°, β=90°, γ=120°, respectively.

The R factor and Rfree of refinement are 0.133 and 0.190 with highly precise phase on 3-D structure of SUMO-2. Comparison of crystal structure between human SUMO-2 and yeast SMT3 shows the r.m.s. deviation of Cα coordinate is  1.054 Å. In addition, comparison of SUMO-1 NMR structure and SMT3 crystal structure shows the r.m.s. deviation of Cα coordinate is 2.736 Å. Hence, the structures of SUMO-2 and SMT3 are more similar than SUMO-1and SMT3.

 

 

PS20

Crystal structure of the chromomycin A3-d(TTGGCCAA)2 complex reveals

GGCC binding specificity of the drug dimer chelated by metal ion.

 

Ming-Hon Hou, Howard Robinson, Yi-Gui Gao and Andrew H.-J. Wang

1Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan and

2National Taiwan University, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

The anticancer antibiotics chromomycin A3 (Chro) is DNA minor groove binding drugs belonging to the aureolic family.  Chro likely exerts its activity by interfering with important biological processes including replication, transcription, and topoisomerization.  Chro forms a dimer mediated by a divalent metal ion which binds to G/C rich DNA.  Herein, we report the first crystal structure of Chro bound to d(TTGGCCAA)2 DNA duplex at 2.15Å resolution solved by the multiwavelength anomalous diffraction (MAD) based on the Co(III) ion.  The crystal structure in the asymmetric unit contains two complexes of metal-coordinated dimers of Chro bound to the octamer DNA duplex.  DNA is kinked by 30° and 36° in the two complexes, individually.  The metal ion is octahedrally coordinated to the O1 and O9 oxygen atoms of the chromophore and two water molecules act as the fifth and sixth ligands. The two coordinated water molecules are hydrogens bonded to O2 atoms of C5 and C13.  Four G-specific hydrogen bonds between Chro and DNA (G4/G12 N2-ChroO8 and G3/G11 N2-Chro EO1’) provide the GGCC sequence specificity for Chro.  Interestingly, DNA appears to act as an effective template to catalyze the deamination of Co(NH3)63+ as shown by crystal data.  In summary, our results present useful structural information for designing new anticancer drug derivatives, as described in our accompanying paper.

 

  

PS21

Design and characterization of a multimeric DNA binding protein using

Sac7d and GCN4 as templates

 

Sz-Wei Wu1,2, Tzu-Ping Ko2, Chia-Cheng Chou3 and Andrew H.-J. Wang1,2,3

1Institute of Biochemical Sciences, National Taiwan University, 2Institute of Biological Chemistry, and

 3Core Facility for Protein X-ray Crystallography, Academia Sinica, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

The protein Sac7d belongs to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Sac7d is extremely stable to heat, acid and chemical agents. This protein is a momomer and it binds DNA without any particular sequence preference. By appending a leucine-zipper-like helical peptide to the C-terminal end of Sac7d, the monomers are expected to interact with each other via hydrophobic force to form a dimer. Large amount of recombinant Sac7d (S7dLZ) was expressed in Escherichia coli and purified by heating and ion-exchange chromatography. The formation of dimer was detected by gel-filtration chromatography and chemical cross-link via glutaraldehyde. The results of surface plasma resonance and circular dichroism experiments showed that the DNA-binding capacity was retained. Furthermore, X-ray diffraction analysis of single crystals of S7dLZ in complex with DNA showed the leucine-zipper segments of S7dLZ were associated into an antiparallel four-helix bundle, and each tetramer binds two DNA fragments. This model works as a successful template that endows protein new function without losing original properties.

 

 

PS22

Probing the Conformational Change of E. coli Undecaprenyl Pyrophosphate Synthase during Catalysis Using an Inhibitor and Tryptophan Mutants

 

Annie Pei-Chun Chen2, Yi-Kai Chen1, Yi-Hung Chen1, Shiao-Chung Chuang2, Chao-Tsen Chen3, and Po-Huang Liang1,2*

1Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan,

 2Institute of Biochemical Sciences, National Taiwan University, Taipei 10098, Taiwan and

3Department of Chemistry, National Taiwan University, Taipei 10098, Taiwan

 

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation reactions of eight isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C55 undecaprenyl pyrophosphate (UPP) (5). This enzyme is essential for bacterial a survival due to its vital role in cell wall synthesis. From our previous studies, we have examined the multiple-step UPPs kinetics and define its reaction mechanisms as well as its protein conformational changes (1,2,4).  Furthermore, our three-dimensional structure of E.Coli UPPs has revealed an elongated hydrophobic tunnel-shaped crevice, and also demonstrated the role of a flexible loop in catalysis (3). In the present study, 7- (2,6-dimethyl-8-diphospho-2,6-octadienyloxy)-8-methyl-4-trifluoromethyl-chromen-2-one geranyl

pyrophosphate (CPCF3), a highly fluorescent analogue of farnesyl pyrophosphate (FPP), was prepared and utilized to study ligand interactions with E. coli undecaprenyl pyrophosphate synthase (UPPs) (6). It was found to be a competitive inhibitor with respect to FPP (Ki = 0.57μM) and also serves as an alternative substrate (Km = 0.69μM and kcat = 0.02 s-1), but mainly reacts with one isopentenyl pyrophosphate (IPP). The fluorescent analogue of FPP we synthesized may provide a tool to investigate the ligand interactions for a broad class of FPP-binding protein. Moreover, the topology of FPP chain elongation proceeds through A69 and L67 in βB and finally reaches L137 at the end of βD was probed by examining the product distribution synthesized by mutants of UPPs enzyme including L137S, L137G, L137A/V105A, A69W, A69L/A143V, L67W, L65W, I141W, and L139W.

 

 

PS23

Crystal Structure of Octaprenyl Pyrophosphate Synthase from Hyperthermophilic Thermotoga maritima and Mechanism of Product

Chain Length Determination

 

Rey-Ting Guo‡§, Chia-Cheng Chou¶||, Tzu-Ping Ko, Hui-Lin Shr¶||, Po-Huang Liang§¶* and Andrew H.-J. Wang§¶||*

Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan

§Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan

Institute of Biological Chemistry, ||Core Facility for Protein X-ray Crystallography,

 Academia Sinica, Taipei 115, Taiwan

 

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C40 octaprenyl pyrophosphate (OPP) which constitutes the side chain of bacterial ubiquinone or menaquinone. So far the structure of long chain synthesizing trans-polyprenyl pyrophosphate has not been reported. In this study, the first structure of long-chain C40-OPPs from Thermotoga maritima has been determined to 2.28 Å resolution. OPPs is composed entirely of a-helices joined by connecting loops and is arranged with 9 core helices around a large central cavity. An elongated hydrophobic tunnel between D and F a-helices contains DDxxD motifs on the top for substrate binding and is occupied at the bottom with two large residues F52 and F132. The products of the mutant F132A OPPs are predominantly C50, longer than the C40 synthesized by the wild type and F52A mutant OPPs, suggesting that F132 is the key residue for determining the product chain length. A76 and S77 located close to the FPP binding site and V73 positioned further down the tunnel were individually mutated to larger amino acids. A76Y and S77F mainly produce C20 indicating that the mutated large residues in the vicinity of the FPP site limit the substrate chain elongation. A76 is the 5th amino acid upstream from the first DDxxD motif on helix D of OPPs and its corresponding amino acid in FPPs is Tyr. In contrast, V73Y mutation led to additional accumulation of C30 intermediate. The new structure of the trans-type OPPs, together with the recently determined cis-type UPPs, significantly extends our understanding on the biosynthesis of long chain polyprenyl molecules.

 

 

PS24

Studies of the structural determinants in a short β-sheet peptide

 

Nicole N.-W. Kuo1, Joseph J.-T. Huang1, Rita P.-Y. Chen2, Su-Ching Lin1, Sunney I. Chan1

1Institute of Chemistry and 2Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei, Taiwan, R..O.C.

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

  D-form Pro (DP) has widely been used in the turn region in designed peptides.  A short peptide (denoted 20-mer, sequence VFITSDPGKTYTEVDPGOKILQ) has been designed by Stanger and Gellman in 1998 (J. Am. Chem. Soc. 120, 4236-4237) and shown to form a stable triple-stranded β-sheet.  Previous studies in our group has disclosed that replacing D-form Pro with Asp destabilized the peptide (Chen et al. (2001) Protein Science 10, 1794-1800).  Especially, the DP to D mutation at DP-6 position caused one amino-acid frameshift of the first β-strand toward the turn region and resulted in side-chain inversion of the first β-strand and a five-residue TSDGK turn.  However, similar mutation at DP-14 position did not change the hydrogen bond network and formed a VDGO turn. The frameshift in the first hairpin raised the questions: whether the structural rearrangement is driven by the new turn sequence or by the hydrophobic interactions of new strand pairings.  Here, we replaced the TSDGK turn with the VDGO turn in the first hairpin.  Interestingly, the resulting  19-mer peptide showed a even more stable β-sheet structure than the wild-type 20-mer.  Our observation suggests that the original side-chain pairings on 20-mer might be forced to form by the dominating DPG turn, and that frameshift caused by the DP-6 to D mutation is driven by more favored side-chain interactions and the resulting TSDGK turn is the compensative product by protruding a bulge in the turn region.

 

  

PS25

Crystal structure of phytase from Selenomonas ruminantium and its complex with persulfated phytate in two binding states

 

Hsing-Mao Chu 1,2,4*, Rey-Ting Guo 1,2,4, Chia-Cheng Chou1,2, Hui-Lin Shr 1,2, Hui-Lin Lai3,

Tsung-Yin Tang3, Kuo-Joan Cheng3 , Andrew H.-J. Wang1,2,4

1Institute of Biological Chemistry, 2Core Facility for Protein X-ray Crystallography,

3Institute of BioAgricultural Sciences, Academia Sinica, Taipei 115, Taiwan, and

4Institute of Biochemical Sciences, National Taiwan University, Taipei, 106 Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

Inositol polyphosphates (IPP) play important roles in many cellular processes. Various inositide phosphatases participate in the regulation of these IPP signaling molecules. In plants, phytate is found abundantly in seeds serving as inorganic phosphate reservoir. Phytases are phosphatases that hydrolyze phytate to less-phosphorylated myo-inositol derivatives and phosphate. The phytase from Selenomonas ruminantium is active without any metal cofactor, which differs from other microbial enzymes that are calcium-dependent. Comparison of the amino acid sequence of S. ruminantium phytase with those of other phytases did not reveal obvious homology. We solved its crystal structure which revealed a new phytase fold that shares a high similarity with that of dual specificity phosphatases. The active site is located near a cysteine-containing P-loop with a conserved sequence of DX(30)HCXXGXXR(T/S). In addition, we also solved two other crystal forms in which an enzyme inhibitor, myo-inositol hexasulfate (IHS), is co-crystallized with the enzyme. In one “standby” crystal form, the inhibitor is bound in a pocket slightly away from the Cys241 in the active site. In the other “inhibited” crystal form, the inhibitor is bound at the substrate-binding site where the to-be-hydrolyzed phosphate group is held close to the –SH group of Cys241. Our structural and mutagenesis studies offer for the first time the visualization on the way by which the P-loop containing phytase attracts and hydrolyzes the substrate (phytate) sequentially.

 

 

 

PS26

C-Terminal Truncation of Helicobacter pylori Fucosyltransferase Improving the Protein Overproduction, Solubility and Activity

 

Sheng-Wei Lin, Jen-Ru Li, Tsui-Min Yuan, Chun-Hung Lin

Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

Helicobacter pylori is an important human pathogen to cause both gastric and duodenal ulcers. The infection is often associated with the occurrence of gastric cancer and lymphoma. This microorganism can express fucose-containing Lewis x and Lewis y antigens as the specific epitopes presented by cell-surface glycoconjugates for the cell adhesion to gastric epithelium cells. The antigen is also able to prevent the immune surveillance by mimicking the host’s cell-surface carbohydrates. Herein we report the expression of fucosyltransferase (FucT) from H. pylor in E. coli. that contains a tandem repeat of leucine zipper as well as a domain rich of basic residues in the C-terminal. Several mutants of different size were prepared by C-terminal truncation at different length in order to investigate the functional roles played by the two regions and improve the formation of soluble proteins. The results indicated that up to 80 amino acid residues, the more residues were deleted, the higher amount of the soluble form was obtained. All truncated FucTs catalyzed the transfer of fucose from GDP-fucose to N-acetyllactosamine at 5 mole/min/mg. We are currently studying the structural change associated with the C-terminal deletion.

 

 

PS27

Catalytic mechanism revealed by the crystal structure, Identification of the Active Conformation and the Importance of Length of the Flexible

Loop 72-83 in Regulating the Conformational Change in Undecaprenyl Pyrophosphate Synthase

Sing-Yang Chang, Tzu-Ping Ko, Yi-Kai Chen, Po-Huang Liang* and Andrew H.-J. Wang*

1Institute of Biological Chemistry, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate (UPP) via condensation with eight isopentenyl pyrophosphates (IPP). UPPs from Escherichia coli is a dimer, and each subunit consists of 253 amino acid residues. The chain length of the product is modulated by a hydrophobic active site tunnel. In this paper, the crystal structure of E. coli UPPs was refined to 1.73 Å resolution, which showed bound sulfate and magnesium ions as well as Triton X-100 molecules. The amino acid residues 72–82, which encompass an essential catalytic loop not seen in the previous apoenzyme structure (Ko, T.-P., Chen, Y. K., Robinson, H., Tsai, P. C., Gao, Y.-G., Chen, A. P.-C., Wang, A. H.-J., and Liang, P.-H. (2001) J. Biol. Chem. 276, 47474–47482), also became visible in one subunit. The sulfate ions suggest locations of the pyrophosphate groups of FPP and IPP in the active site. The Mg2+ is chelated by His-199 and Glu-213 from different subunits and possibly plays a structural rather than catalytic role. However, the metal ion is near the IPP-binding site, and double mutation of His-199 and Glu-213 to alanines showed a remarkable increase of Km value for IPP. Inside the tunnel, one Triton surrounds the top portion of the tunnel, and the other occupies the bottom part. These two Triton molecules may mimic the hydrocarbon moiety of the UPP product in the active site. On the other hand, in order to identify the active conformation and study the role of the loop for conformational change, the UPPs mutants with amino acids inserted into or deleted from the loop were examined.

 

 

 

PS28

Structural Analysis and Protein Engineering of RD1, a Type III

Antifreeze Protein

 

Ming-Feng Hsu1,2, Tzu-Ping Ko 2, and Andrew H.-J. Wang1,2.

1Institute of Biological Chemistry, Academia Sinica, and 2Institute of Biochemical Sciences,

National Taiwan University.

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

Antifreeze proteins comprise a structurally diverse class of proteins that have in common the ability to bind to ice and inhibit its growth. There are four types of antifreeze proteins, one of which is a glycopeptide. Interestingly, according to the result of sequence alignment, the C-terminal region of mammalian sialic acid synthase is homologous to fish type III antifreeze protein. The highest similarity between these two proteins is in the protein core and the flat ice-binding regions. The gene of RD1, a 7 kDa type III antifreeze protein from Antarctic eel pout, was cloned and expressed in Escherichia coli. A synthetic gene for RD1 using the preferred codons of E. coli was inserted into pET16b and expressed in BL21(DE3). The recombinant wild type RD1 was expressed at 16 and induced for 30 hours, and mutants of RD1 had been refolded. Crystals of RD1 were obtained by the hanging-drop vapor-diffusion method. RD1 crystals belong to the orthorthombic space group P212121, with unit-cell parameters a = 32.0, b = 39.7, c = 45.0 Å, and diffract to 1.5 Å resolution. There is a hydrophobic cavity in the RD1 molecule, with a volume of 45 Å3 and surrounded by eight conserved non-polar residues: Val5, Ala7, Ile11, Leu17, Met 21, Met22, Val49, and Leu55. We designed several point-mutated proteins, including V49A, V49L, V49W, and L55A, L55V, L55W, to study the stability and folding of RD1. We also fund that RD1 is homologous to the C-terminal region of sialic acid synthase. Similarity is greatest in the protein core and the flat ice-binding region. Streptoccus alage NeuB is also called sialic acid synthase, and we already got the crystals of NeuB.

 

  

PS29

Structures and Disulfide Bonds of Venom Phospholipases A2­­ in Two Primitive Tree Vipers: Trimeresurus puniceus and Trimeresurus borneenesis

 

Y. M. Wang*, H. F. Peng and I. H. Tsai

Inst. Biol. Chemistry, Academia Sinica; Ins. Biochem. Sciences,

National Taiwan University, P. O. Box 23-106, Taipei, Taiwan, R.O.C.

 

By gel filtration and reversed-phase HPLC, phospholipase A­2 (PLA­) isoforms were purified from venoms of Indonesian tree vipers T. puniceus and T. borneenesis. By mtDNA phylogeny, both species were shown to be primitive within this genus. The enzymatic activities, N-terminal sequences and precise masses of the PLAs were determined. Using the cDNA prepared from the venom glands, three Lys49-PLAs as well as several basic and acidic Asp49-PLAs were cloned and sequenced. The masses and the N-terminal sequences of the purified PLAs from both venom species were matched successfully to each of the cDNA-deduced amino acid sequences. Notably, each of the Lys49-PLA contains only six disulfide bridges and lack the C61-C91 normally conserved in other pit viper venom PLAs so far studied. In contrast, all the venom Asp49-PLAs in these tree vipers contain seven pairs of disulfides per molecule. With hardly detectable enzymatic activity, the Lys49-PLAs induced edema on the rat paws. Their 3D structure models are almost identical to the  template crystallographic structure of Lys49-PLA from Ce. godmani venom except the region near position 61. However, they are less stable than the seven-disulfide-containing Lys49-PLAs from T. stejnegeri venom, as indicated by a difference of 8.8 °C in the melting temperature, as monitored by the circular dichroism spectroscopy. The relationships of the venom Lys49-PLAs were studied by phylogenetic analyses based on their amino acid sequences and a close link between the T. puniceus and T. stejnegeri Lys49-PLAs was confirmed.  

 

 

PS30

Biophysical and Biochemical Characterization on the Quaternary Structure of Sialic Acid Synthase

 

Tzann-Shun Hwang1,2*, T. Booma Kasthuri1,3, Chin Fen Teo1, Xing-Hung Hwang3, Yu-Ju Chen3, Chun-Hung Lin1,2

1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

2Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan

3Institute of Chemistry, Academia Sinica, Taipei, Taiwan

 

Sialic acid is known as an important virulent determinant present in the capsular polysaccharides of pathogenic bacteria associated with infectious diseases. Sialic acid synthase (NeuB), encoded by neuB gene, catalyzes the condensation of N-acetylmannosamine and phospho(enol)pyruvate to form sialic acid in some Gram(-) bacteria (e.g. Escherichia coli) and Gram(+) bacteria (e.g. Streptococcus agalactiae). E. coli NeuB (EcNeuB) and S. agalactiae NeuB (SaNeuB) were expressed and purified to give > 95% homogeneity for further characterization. MALDI-TOF, equilibrium sedimentation and chemical cross-linking analyses indicated that both enzymes existed as a tetrameric form. The studies of pH, temperature, and stability showed that these two enzymes only have a slight difference in the optimal conditions. The pH effect on the quaternary structure was investigated by nanoflow ESI-MS and velocity sedimentation analyses. These data indicated that the tetrameric structures of both enzymes were stable at pH 7.5 - 8.5 and the activity of both enzymes was retained at the same pH range. The circular dichroism analyses revealed that EcNeuB have a higher percentage of a-helix than SaNeuB have. The kinetic parameters of the Km (ManNAc), Km (PEP), and kcat were measured to be 77.17 mM, 5.24 mM and 17.24 min-1 for EcNeuB and 31.14 mM, 1.53 mM and 1.22 min-1 for SaNeuB, respectively. The Lineweaver-Burk plot suggested that the enzyme catalysis of EcNeuB and SaNeuB is an ordered reaction.

 

 

PS31

Role of the Cysteine Residues in Sialic Acid Synthase

 

Jui-Chuan Chen*, Tzann-Shun Hwang, Chiung-Fang Chang, Chun-Hung Lin

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan

 

N-acetylneuraminic acid synthase (NeuB, E.C 4.1.3.19) catalyzes the aldol condensation of phospho(enol)pyruvate (PEP) with N-acetylmannosamine (ManNAc) to form sialic acid in both E. coli K1 and Streptococcus agalactiae. Both recombinant enzymes were purified and inactivated by several sulfhydryl-modifying reagents including 5,5-dithiobis-2-nitrobenzoate (DTNB), N-ethylmaleimide (NEM), iodoacetic acid (IAA), methyl methane thiosulphonate (MMTS), iodoacetamide (IAM) and 2-nitro-5-thiocyanobenzoic acid (NTCB). DTNB was used to carry out the titration of sulfhydryl group. No disulfide bond was found in both NeuB proteins indicating that the inactivation is not caused by the disruption of disulfide bond. Chemical modification and substrate protection were performed to elucidate the catalytic role of cysteine residues. IAA was observed to inactivate both NeuB proteins in a time- and dose-dependent manner. The substrate protection was effective only in the presence of Mn2+ and PEP.  Four mutants (corresponding to the NeuB from E. coli K1) were generated by site-directed mutagenesis CysAla. Kinetics studies showed that no significant changes were observed for the NeuB mutants including C176A, C255A and C260A. Nevertheless, the mutant C12A has about 50% activity with increased Km. These results implied that Cys12 plays an important role in catalysis.

 

 

PS32

Crystallization, and Preliminary X-ray Analysis of L-aspartate beta-decarboxylase (Alcaligenes faecalis) from Escherichia coli

 

 

Hui-ju Chen*a,b, Pei-Chien Tsaic, Nai-Chen Wangc, Tzu-Ping Koa, Chia-Yin Leec, and Andrew H.-J. Wanga,b

aTaiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan

bInstitute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan

cGraduate institute of Agricultural Chemistry of National Taiwan University, Taipei 106, Taiwan

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

L-Aspartate B-decarboxylase (Asd ; E.C. 4.1.1.12) catalyses the removal of the beta carboxyl group from L-aspartate with complete retention of the asymmetric centre to produce L-alanine. It is an important enzyme to produce L-alanin and D-aspartate. The recombinant Asd from Alcaligenes faecalis CCRC 11585 of 533 amino acids, with a calculated molecular weight of 59584 Da and a pI of 5.14. Base on the sequence the asdA gene product was overproduced in Escherichia Coli and purified to homogeneity. Crystallographic studies of asdA were initiated in order to study the relationship of the assembly structure and the mechanism of D-amino acid produced. By the hang-in vapor-diffusion method, the protein crystallized using 0.1M Tris pH 7.0-7.5, 0.1M LiSO4, 20% PEG4000 at 277K for 30 days to 10 month. Preliminary X-ray analysis showed that the crystal belongs to space group C222, with unit-cell parameters a=150.7, b=217.2, c=209.4 Å and six molecule per asymmetric unit. A comparable 2.7Å data set was measured at 180K using a synchrotron of NSRRC source and Raxis IV imaging plate area detector (96.6% completeness, Rmerge = 10.7%). Molecular replacement studies and multiple anomalous dispersion (MAD) experiments are currently in progress in order to determine the detailed three-dimensional structure of asdA.

 

  

PS33

Biophysical Characteristics of Cytotoxic Ribonucleases from

Rana catesbeiana

Yuan-Chao Lou1, Chun-Hua Hsu2, Yun-Ru Pan1, Yi-Hsuan Ho1, Chung-De Chen3, You-Di Liao1, Chinpan Chen1*

1Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 3Department of Physics, Fu-Jen University, Taipei, Taiwan

No. 128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

Bullfrog ribonucleases from Rana catesbeiana are found to exhibit cytotoxicities toward tumor cells in addition to possessing different base specificities and ribonucleolytic activities. Therefore, understanding the structure/function relationships of these enzymes would be helpful toward investigating whether they have potential for use as agents in tumor therapy. We have expressed and purified several frog ribonucleases as well as mutant proteins. With good stability and well-dispersed NMR data, these proteins are superb systems for NMR structural studies as well as for other biophysical studies. CD and NMR data clearly reveal that these frog ribonucleases possess highly similar secondary structures. Based on the comparison between native and recombinant protein structures, we concluded that the reduction in catalytic and cytotoxic activities for the recombinant protein, which contains an extra Met residue and has a Gln instead of pyroglutamate at the N-terminus, is primarily due to the loss of two H-bonds in the N-terminal Gln residue. Mutational studies of RC-RNase 4 and RC-RNase 2 clearly indicated that Trp15 plays an important role in high thermostability and causes a unique characteristic of CD data with an ellipticity minimum at 228 nm for RC-RNase 4. NMR chemical shift perturbations between free- and complex- structures identified the substrate-bound related residues in the base specificity for the frog ribonucleases. In addition, the complex structure of RC-RNase L1 with d(ApCpGpA) substrate analog with also be discussed.

 

 

PS34

Automatic Discovery of Recurrent Local Structures for Approximating Three-Dimensional Protein Molecules

 

Ta-Tsen Soong1,2,*, Ming-Jing Hwang1 and Chung-Ming Chen2

1Institute of Biomedical Sciences, Academia Sinica, Nan-Kang, Taipei, Taiwan and
2Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan

 

The scope of conformation space the protein molecules can adopt is a problem of main interest. Previous studies in other groups have shown that there are stereochemical constraints that confine local protein structures to a limited range of conformations. Furthermore, the results of many groups have demonstrated that the sequence-to-structure relationship remains detectable on a local level. By studying the conformational space of local protein structures, we will obtain more information concerning the constraints on local space and sequence-to-structure mapping; hence facilitate ab initio structure prediction. In this study, we propose a novel algorithm that automatically discovers recurrent pentamer structures in proteins.

The algorithm starts by applying Expectation-Maximization (EM) clustering to the distances between non-adjacent backbone Cα atoms in a large set of pentamer fragments. A rough partition of the conformation space can thus be derived. In the second stage, by applying a split-and-merge algorithm, we can obtain a finite number of clusters and guarantee the homogeneity and distinctiveness of each one. The results show that, with 41 major representative structures, we can approximate most of the protein fragments with an error of 0.378 Å. With only 20 types of structures, the fragment structures can still be modeled at 0.44 Å, which is comparable to or better than the performance of previous methods. We term the representatives as “building blocks”. At the global level, we demonstrate that by concatenating different combinations of building blocks, we can model whole protein structures at high resolution. A resolution of 2.54 Å can be achieved simply by using 10 types of building blocks. This finding suggests that the study of molecular structures can be hugely simplified using this reduced expression.

 

  

PS35

Conformational Change of GroEL studied by Tyrosine Fluorescence

 

Kazuhiko Hosono1*, Taro Ueno1, Hideki Taguchi2, Fumihiro Motojima2, Tamotsu Zako1,

Masasuke Yoshida2 and Takashi Funatsu1

1 Department of Physics, School of Science and Engineering, Waseda University

2 Chemical Resources Laboratory, Tokyo Institute of Technology

1 3-4-1 Ohkubo, Shinjuku 169-8555 Tokyo Japan, 2 4259 Nagatsuta, Yokohama 226-8503, Japan.

 

GroEL, an E. coli chaperonin, undergoes a cycle of repetitive binding and release of GroES with the energy of hydrolysis of ATP. During this cycle, it mediates protein folding in its central cavity. Although conformational differences of GroEL with or without GroES were studied by X-ray crystallography, dynamics of conformational changes during the chaperonin cycle remained unclear.  We performed real time detection of the conformational change of GroEL in the absence of denatured protein by measuring the change in fluorescence intensity of tyrosine with a fluorometer equipped with a stopped flow system. A solution containing GroEL and GroES was mixed with a solution containing ATP to start the chaperonin reaction cycle. Fluorescence intensity of tyrosine increased to 124% with rate constant of 0.45s-1, then followed by decrease to the steady level of 113%. We also performed similar experiments using D398A, ATP deficient mutant, or SR1, single ring version of GroEL which stops the cycle right before ATP hydrolysis. In this case, only an increase followed by a steady level with no decrease in fluorescence intensity was observed. These results suggested that conformational changes of GroEL occurs at rate constant of 0.48s-1 after binding of GroES, then followed by another conformational change with 0.44s-1 which was followed by hydrolysis of ATP. From other experiments using GroEL mutants (Y360F, Y476F, Y478F, Y485F, Y506E, Y506W), it was revealed that Y506 is responsible for the change in fluorescence intensity. This tyrosine residue is located near the binding site of nucleotide. Therefore the signal may reflect the change of nucleotide states.

 

 

PS36

The Study of the Interaction between Transthyretin and

Amyloid b-Protein by NMR

 

Atsushi Matsuura*, Mineyuki Mizuguchi, Takahide Kono, Kimiaki Matsubara and Keiichi Kawano

Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University

2630 Sugitani, Toyama 930-0194, Japan

 

The deposition of amyloid b-peptide (Ab) is the cardinal pathological feature of the Alzheimer’s disease (AD) brain, and the reduction of Ab is considered as a primary therapeutic target.  The Ab is a 39- to 43-amino acid peptide that is cleaved from amyloid precursor protein (APP) by two sequential proteolytic processes with b- and g-secretases Transthyretin (TTR) is a protein synthesized independently and primarily in the liver and choroid plexus of the brain, which circulates in plasma and CSF as a 55-kDa tetramer composed of 127-amino acid monomers.  A number of recent studies have suggested that TTR plays an important role in the pathogenesis of AD.  Therefore, knowing how the Ab clearance and deposition are influenced by TTR is important for understanding the pathology of AD and developing ways to design potential AD therapeutics.  In the present study, we report the inhibition of Ab fibrillogenesis by TTR and the identification of the binding interface between Ab and TTR.  We examined the effect of TTR on aggregation of the 40- and 42-amino acid Ab (Ab1-40 and Ab1-42) using a quantitative thioflavin-T fluorometric assay in vitro.  Our data showed that TTR prevents amyloid fibril formation of Ab1-40 and Ab1-42 Furthermore, we successfully identified the Ab-binding surface on TTR by monitoring the Ab-induced perturbations of NMR resonances from 2H/15N-labeled TTR.

 

  

PS37

Structural Analysis of Membrane-Bound Mastoparan-X by Solid-State NMR

 

Yasuto Todokoro1*, Hajime Yanagishita2, Kang. S. W.3, Toshimichi Fujiwara1and Hideo Akutsu1

1Institute for Protein Research, Osaka University, Yamadaoka, Suita, 565-0871, Japan,

2Yokohama National University, Japan and 3Pusan National University, Korea

 

Mastoparan is amphiphilic peptide isolated from wasp venom which activate GTP-binding regulatory protein (G protein). The conformation of Mastoparan-X (MP-X) bound to membrane was reported to be an α-helix from Trp3 to C-terminal Leu14, while it is a random coil in water. The bound state conformation was determined by liquid-state transferred nuclear Overhauser effect (TRNOE) NMR. It is essentially an indirect method. In order to directly determine its bound conformation, solid-state NMR should be useful. Here we conducted the structural analysis of membrane-bound Mastoparan-X by Solid-State NMR. We determined the secondary structure of Mastoparan-X strongly bound to the phospholipid bilayers by precise distance measurements and torsion angle prediction using TALOS (Torsion Angle Likelihood Obtained from Shifts and sequence similarity). Distance measurements were used by constant time rotational resonance and rotational echo double resonance (REDOR) solid-state NMR experiments. The constant time is important for the membrane bound system. The rotational resonance method gave 4.45±0.01Å, 4.57±0.04Å for the distances between 1-13C of Gly5 and 3-13C of Ala8, and between 1-13C of Ala7 and 3-13C of Ala10 respectively. The REDOR gave 4.59±0.06Å, 4.92±0.06Å for the distances between 1-13C of Ala10 and 15N of Leu13, and between 1-13C of Ala10 and 15N of Leu14 respectively. Also, 13C and 15N backbone of uniformly labeled MPX was assigned by intra-residue C-C and N-Cα correlation experiments and inter-residue Cα-Cα, N-Cα and N-C’-Cα correlation experiments. By structural calculation using these four distance restraints and 24 dihedral angle restrains derived from chemical shifts by TALOS, Membrane bound MP-X was found to have an α-helical conformation from Trp3 to Leu13, which was similar to an α-helical conformation determined by TRNOE.

 

 

PS38

NMR Investigation on the Interaction of Cytochrome c3 with its

Physiological Partner [NiFe] Hydrogenase

Naoki Yahata1*, Takashi Saitoh1, Asuka Nakahara2, Hideaki Ogata2, Yoshiki Higuchi2, and Hideo Akutsu1

1Institute for Protein Research, Osaka University, Japan and 2Graduate School of Science, Himeji Institute of Technology, Japan, 3-2 Yamadaoka, Suita, Osaka 565-0871 Japan

 

Cytochrome c3 isolated from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F, is a tetraheme protein. Its physiological partner, [NiFe] hydrogenase, catalyzes the reversible oxidoreduction of molecular hydrogen. These two proteins, which play central roles in hydrogen metabolism, form a transient complex for exchanging electrons. To elucidate the electron transfer mechanism between cytochrome c3 and [NiFe] hydrogenase, heteronuclear NMR experiments were performed. 1H-15N HSQC spectra were measured for 15N labeled ferric and ferrous cytochrome c3, in the absence and in the presence of unlabeled [NiFe] hydrogenase. Using these data, chemical shift perturbation mappings were performed for ferric and ferrous types, respectively.

For ferric cytochrome c3, chemical shift changes were observed in the region around heme 4. Cytochrome c3 is a basic protein and possesses many lysine residues around heme 4. In contrast, [NiFe] hydrogenase is an acidic protein. So this result is reasonable from the electrostatic point of view. On the other hand, the chemical shift perturbation pattern of the ferrous cytochrome c3 was different from that of ferric one. In addition to the residues around heme 4, those around heme 3 were affected in the presence of [NiFe] hydrogenase. The different modes of interactions in different oxidation states will be discussed in connection with its physiological functions.

 

 

PS39

Role of flexible N-terminal residues in biological activity of the insect cytokine, growth blocking peptide (GBP)

Masanobu Yoshida1*, Kunio Shitara1, Kimiaki Matsubara1, Takahide Kouno1, Tomoyasu Aizawa2, Yoichi Hayakawa3,

Yasuhiro Kumaki2, Mineyuki Mizuguchi1, Makoto Demura2, Katsutoshi Nitta2, and Keiichi Kawano1

1Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan,

2Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan, and 3Institute of Low Temperature Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0819, Japan

 

Growth-blocking peptide (GBP) is a 25 amino acid cytokine isolated from Lepidopteran insects that exhibits various biological activities, such as regulation of larval growth of Lepidopteran insects, proliferation of a few kinds of cultured cells and stimulation of a class of insect immune cells (referred to as plasmatocyte).  The tertiary structure of GBP consists of a well-structured core domain and disordered N and C termini.  Our previous studies revealed that not only the core structure of GBP but the particular residues in the unstructured N-terminal region, such as Glu1 and Phe3, were also essential for the plasmatocyte-stimulating activity.  In this study, a number of deletion, insertion and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and receptor.  Alteration of the backbone length of linker region between the core structure and N-terminal domain reduced the plasmatocyte-stimulating activity.  The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity.  We conclude that the receptor interaction of GBP depends upon the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding.  Additionally, antagonistic experiments using deletion mutants confirmed that both the core and the N-terminal domain of GBP were required for receptor binding, and furthermore Phe3 is a binding determinant of the N-terminal domain. 

 

 

PS40

Effects of the amino acid substitutions in the DE loop of transthyretin on the protein structure and amyloid fibril formation

Makoto Takeuchi*, Mineyuki Mizuguchi, Atsushi Matsuura, Kimiaki Matsubara and Keiichi Kawano

Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University

2630 Sugitani, Toyama 930-0194, Japan

 

Amyloidosis is a group of diseases characterized by local or systemic extracellular deposits of insoluble amyloid fibrils.  Amyloid fibrils of transthyretin (TTR) are known to cause familial amyloid polyneuropathy (FAP).  Over 80 different mutations in TTR are known, and most of them are associated with FAP.  The prevailing hypothesis for the property of TTR mutants to form fibrils is that the FAP mutations affect the stability of the native tetrameric TTR and facilitate its conformational change to amyloidogenic intermediates, which self-assemble into amyloid fibrils.  In this study, the structure and stability of the FAP mutants of TTR (L58H, L58R, T59K, E61K) were compared with those of wild-type TTR.  In these FAP mutants, the amino acid residue in the loop between the D- and E-stand is substituted with the basic amino acid.  The tetramer to monomer equilibrium of the L58H, L58R, T59K and E61K TTR was analyzed by SDS-PAGE, indicating that the quaternary structure of these mutants is destabilized compared with that of wild-type TTR.  Thioflavin T binding to amyloid fibrils was performed to investigate the extent of amyloid fibril formation of the wild-type and mutant TTRs.  The L58H, L58R, T59K and E61K TTR formed more amyloid fibrils than the wild-type TTR, showing that the FAP mutations in the DE loop increase amyloidogenicity of TTR.  We also investigated the stabilities of wild-type and mutant TTRs by using circular dichroism and differential scanning calorimetry.  All of the mutants in this study showed the reduced structural stability compared with the wild-type TTR.  We will report the proton-deuterium exchange data by NMR spectroscopy for the wild-type and the mutant TTRs and discuss the relationship between the amyloidogenicity and the stability of TTR structure.

 

  

PS41

Possible Involvement of a FKBP family Peptidyl-prolyl cis-trans Isomerase from Psychrotrophic Bacterium Shewanella sp. SIB1 in Cold-Adaptation

Yutaka Suzuki1*, Mitsuru Haruki 2 Kazufumi Takano1 Masaaki Morikawa1 and Shigenori Kanaya1

1Department of Material and Life Science, Graduate School of Engineering, Osaka University and

 2Department of Materials Chemistry and Engineering, College of Engineering,

Nihon University2-1, Yamadaoka, Suita, Osaka

 

Using the psychotrophic bacterium Shewanella sp. SIB1, which grows at low temperature, we observed by protein separation in 2D-PAGE that some proteins are expressed at elevated levels at low temperature. One in this respect particularly prominent protein had a molecular mass around 28,000, and partial sequence analysis revealed that it belongs to the family of peptidyl-prolyl cis-trans isomerases. Such proteins are present in all species and appear to be involved in proper protein folding although elimination of members of the group often has no recognizable effect on the organism. This led us to suspect that the cold induction of the enzyme in Shewanella might reveal something about the function of this protein. We therefore cloned the gene, expressed the protein in E. coli, purified it and analyzed several of its enzymatic properties. In one of the assays refolding of RNase T1 was determined at lower and higher temperature in the absence and in the presence of the enzyme. While at higher temperature, the kinetics of renaturation was not affected by the enzyme, at lower temperature the enzyme significantly increased the rate of renaturation over the spontaneous one. In the other assay, we examined the significance of its dimeric structure by constructing a mutant in which its dimerization core was truncated. This revealed that the dimeric structure is necessary to accelerate the rate of RNase T1 refolding, although the protein in the monomeric sturucture can efficienty catalyze the prolyl-isomerization of tetra-peptides. Thus it appears that this enzyme, and possibly other peptidyl-prolyl cis-trans isomerases as well, may be involved in protein folding under low temperature at which the spontaneous rate of refolding is particularly low.

*This work was supported in part by a research grant from the Kurita Water and Environment Foundation (KWEF)

 

 

PS42

Cross-Linking Effect Caused by Local Stabilization of the Double Helix

Dmitri Lando 1,2 *, Alexander Fridman 2 and Valentina P. Egorova1,2

1 Institute of Botany, Academia Sinica, Nankang, Taipei, 11529, Taiwan, and 2 Institute of Bioorganic Chemistry,

Belarus National Academy of Sciences, Kuprevich St., 5/2, 220141 Minsk, Belarus

 

Usually DNA interstrand cross-links are formed due to bidentate covalent binding of platinum antitumor compounds and some other drugs to nucleotides of different strands. In this study, we have found that other types of chemical modifications (intrastrand cross-links or monofunctional adducts) also caused by these compounds can give rise to interstrand linkage if they stabilize the double helix at sites of modification strongly. This local stabilization makes DNA melting fully reversible and independent of strand concentration as it is inherent to ordinary covalent interstrand cross-links. Our studies show that an increase by 25¸30 kcal in the free energy of the helix-coil transition of a single base pair in a long DNA chain is sufficient for “cross-linking effect”, i.e. for preventing strand separation after melting of ordinary (nonmodified) AT and GC base pairs. If this strong stabilization is distributed along several neighboring base pairs, then much lower minimum increase per stabilized base pair is sufficient for appearance of such a “cross-linking effect”. Besides covalent binding of antitumor compounds, relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (more than 20‑40bp) can cause the cross-linking.

This work was supported by Fund of Fundamental Investigations of the Republic of Belarus (X02R-049) and by ISTC Foundation (A301.2).

 

 

PS43

Role of flexible N-terminal residues in biological activity of the insect cytokine, growth blocking peptide (GBP) 

Masanobu Yoshida1*, Kunio Shitara1, Kimiaki Matsubara1, Takahide Kouno1, Tomoyasu Aizawa2, Yoichi Hayakawa3,

Yasuhiro Kumaki2, Mineyuki Mizuguchi1, Makoto Demura2, Katsutoshi Nitta2, and Keiichi Kawano1

1Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan,

2Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan, and 3Institute of Low Temperature Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0819, Japan

 

Growth-blocking peptide (GBP) is a 25 amino acid cytokine isolated from Lepidopteran insects that exhibits various biological activities, such as regulation of larval growth of Lepidopteran insects, proliferation of a few kinds of cultured cells and stimulation of a class of insect immune cells (referred to as plasmatocyte).  The tertiary structure of GBP consists of a well-structured core domain and disordered N and C termini.  Our previous studies revealed that not only the core structure of GBP but the particular residues in the unstructured N-terminal region, such as Glu1 and Phe3, were also essential for the plasmatocyte-stimulating activity.  In this study, a number of deletion, insertion and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and receptor.  Alteration of the backbone length of linker region between the core structure and N-terminal domain reduced the plasmatocyte-stimulating activity.  The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity.  We conclude that the receptor interaction of GBP depends upon the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding.  Additionally, antagonistic experiments using deletion mutants confirmed that both the core and the N-terminal domain of GBP were required for receptor binding, and furthermore Phe3 is a binding determinant of the N-terminal domain. 

 

 

PS44

Structure-function relationship of gaegurin 5, an antimicrobial peptide isolated from a Korean frog

Seo-Jeong Jung*, Hyung-Sik Won, Sang-Ho Park, and Bong-Jin Lee

College of Pharmacy, Seoul National University, Seoul 151-742, Korea

San 56-1, Shillim-Dong, Kwanak-Gu, Seoul, 151-742, Korea

 

Gaegurin 5 (GGN5) is a cationic 24-residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. It contains a central proline residue and an intraresidue disulfide bridge in its C-terminus, which are common to the antimicrobial peptides found in Ranidae. We determined the solution structure of GGN5 bound to sodium dodecyl sulfate micelles for the first time and investigated the role of proline, cysteine, and a disulfide bridge on the structure and activity of GGN5. GGN5 adopts an amphipathic aa-helical structure spanning residues 3-20 kinked around Pro14 which affords the hydrophobic residues to reside in the concave helical region, and a disulfide-bridged looplike conformation in its C-terminus. By replacement of proline with alanine (PAGGN5), a straight and rigid helix was formed in the central region and was more stable than the kinked helix. Reduction of a disulfide bridge in C-terminus (GGN5SH) maintained the loosely ordered looplike conformation, while the replacement of two cysteines with serines (CSGGN5) caused the C-terminal conformation completely disordered. The magnitude of antimicrobial activity of the peptides was closely related with the helical stability of them in the order of PAGGN5 > GGN5 > GGN5SH > CSGGN5, suggesting that the helical stability of the peptides is important for the antimicrobial activity. On the other hand, the significant increase of hemolytic activity of PAGGN5 implies that a helical kink of GGN5 could be involved in the selectivity of target cells. The location of GGN5 and PAGGN5, analyzed using paramagnetic probes, was mainly at the surface of SDS micelles, though the location of N-terminal region was slightly different between them.

 

  

PS45

Structure-function relationship of peptide analogues of gaegurin 4, an antimicrobial peptide isolated from a Korean frog

Min-Duk Seo*, Hyung-Sik Won, and Bong-Jin Lee

College of Pharmacy, Seoul National University, Seoul 151-742, Korea

San 56-1, Shillim-Dong, Kwanak-Gu, Seoul, 151-742, Korea

 

Gaegurin 4 (GGN4), a 37-residue antimicrobial peptide, consists of two amphipathic a-helices (residues 2-10 and 16-32) connected by a flexible loop region (residues 11-15). As part of an effort to develop new peptide antibiotics with low molecular mass, the activities of C-terminally truncated GGN4 analogues were tested.  D24-37 GGN4, a peptide analogue with 14 residues truncated from the C-terminus of GGN4, showed a complete loss of antimicrobial activity. However, the single substitution of aspartic acid 16 by tryptophan (D16W) in the D24-37 GGN4 completely restored the antimicrobial activity, without any significant hemolytic activity. In contrast, neither the D16F nor K15W substitution of the D24-37 GGN4 allowed such a dramatic recovery of activity. In addition, the D16W substitution of the native GGN4 significantly enhanced the hemolytic activity as well as the antimicrobial activity. The structural effect of the D16W substitution in the D24-37 GGN4 was investigated by CD, NMR, and fluorescence spectroscopy. The results showed that the single tryptophanyl substitution at position 16 of the D24-37 GGN4 induced an a-helical conformation in the previously flexible loop region in intact GGN4, thereby forming an entirely amphipathic a-helix. In addition, the substituted tryptophan itself plays an important role in the membrane-interaction of the peptide. The present structural investigations of the GGN4 analogues not only contribute to a better understanding of the structure-activity relationships of this group of antimicrobial peptides with a linear amphipathic a-helix, but also suggest that the D16W- D24-37 GGN4 could be considered as a potential target molecule for new peptide antibiotics. In addition, the utility of a tryptophan insertion is proposed for peptide engineering to enhance the helical propensity and/or membrane-interacting ability.

 

 

PS46

Active Site Structure of Ni-Containing Superoxide Dismutase (Ni-SOD)

Jin-Won Lee,1 Tran-Chen Yang,2 Hong-In Lee,3,* Sa-Ouk Kang,1 and Brian M. Hoffman2

1Department of Microbiology, College of Natural Science and Research Center for Microbiology, Seoul National University, Seoul, 151-742, Korea, 2Department of Chemistry, Northwestern University, Evanston,

Illinois 60208, USA and 3Department of Chemistry Education, Kyungpook National University, Daegu, 702-701, Korea

 

Superoxide dismutases (SODs) are the first in biological protection mechanisms against the oxidative stress by catalyzing the disproportionation of superoxide (O2•-) to hydrogen peroxide (H2O2) and molecular oxygen (O2). SODs have been classified by their redox-active metal contents: CuZn-SOD, Mn-SOD, and Fe-SOD. Recently, Ni-containing SOD (Ni-SOD) was isolated from Streptomyces species. The finding of Ni-SOD is of very interest because Cu, Fe, and Mn ions can dismutase superoxide anion in aqueous solution but Ni ion does not, proposing that Ni-SOD is a new class of SODs and the 3-D coordination environments of the metallic site play a key role for both fine tuning of redox potential of the site and enzymatic mechanism. EPR study of the enzyme revealed that the resting-state Ni-SOD contains NI(III) ion in its active site. EXAFS studies of Ni-SOD have proposed two structural models of the Ni site: a five-coordinated mononuclear Ni center with two N(O)- and three S-donor ligands, and a dinuclear Ni-center bridged by two m-cysteinatos with additional two N(O)- and one S-donor ligands to each Ni ion. Such kinds of Ni site structure have not been found among the known redox-active Ni-containing catalytic enzymes, indicating that Ni-SOD is completely a new class in redox-active Ni-containing enzymes, and the coordination and geometrical structure of the site is the key to specifically control the dismutase activity of the enzyme. In the present study, we refine the active-site structure of Ni-SOD by multifrequency advanced EPR spectroscopies.

 

 

PS47

Theoretical studies of protein-folding thermodynamics
and kinetics - A Mean Field Theory

 

Kuo Kan Liang1, Michitoshi Hayashi2, YingJen Shiu1, Yu-Lin Yeh1, C. H. Chang1 and Sheng Hsieh Lin1,2

1Institute of Atomic and Molecular Sciences, Academia Sinica,  Taipei 11529, Taiwan

2Center for Condensed Matter Sciences, National Taiwan University, Taiwan

Institute of Atomic and Molecular Sciences,P.O. Box 23-166, 106, Taipei, Taiwan

 

With regarding a protein as a topological collection of interacting peptide bonds, we applied the kinetic Ising model in the mean field approximation to study the equilibrium and kinetic behaviors of protein folding-unfolding. According to this model, thermodynamics and kinetics of protein folding-unfolding are related to the elementary process of folding 1 unfolding of such interacting units. In the previous two-state model case, the kinetic behavior is predicted to be exponential. Our model can treat the effect of temperature and denaturant concentration on the thermodynamics and kinetics of protein folding-unfolding and provide the chevron plot. Our model can also predict the non-exponential kinetic behavior as consisted with experimental results. This model also predicted the universal curves existing separately for the thermodynamic behaviors and kinetics behaviors of protein folding-unfolding. A further prediction of force-extension curves involved in tweezers and atomic force microscopic experiments will also be presented.

 

 

PS48

Preliminary X-ray Diffraction Analysis of Hydantoinase from Pig Liver and Agrobacterium radiobacter

 

Jia-Yin Tsai1*, Sheng-Kuo Chiang1, Cheng-Yang Huang2, Yuh-Shyong Yang2, Yuh-Ju Sun1

1Institute of Bioinformatics and Structural Biology & Department of Life Science,

 National Tsing Hua University, Hsinchu, Taiwan 300, ROC

2Department of Biological Science and Technology, College of Science,

National Chiao Tung University, Hsinchu, Taiwan 300, ROC

 

Mammalian hydantoinase is the enzyme involved in uracil and thymine catabolism. A thermophilic hydantoinase from pig was purified and crystallized. One belongs to the triclinic P1 space group, with unit-cell parameters a = 96.35, b = 96.87, c = 154.87 Å, a = 82.10, b = 72.54, g = 77.19, and the other belongs to the orthorhombic C2221 space group, with unit-cell parameters a = 113.92, b = 157.22, c = 156.21Å. Bacterial hydantoinase is a zinc enzyme and used in industry as a biocatalyst for the production of D- or L-amino acids. A D-hydantoinase from Agrobacterium radiobacter was expressed in Escherichia coli, purified to homogeneity and crystallized. D-Hydantoinase was crystallized using ammonium sulfate as a precipitant. The crystal form belongs to tetragonal I4 space group, with unit-cell parameters a = b = 129.39Å, c = 173.31Å and diffracted to 2.6Å containing three molecule per asymmetric unit. Taking advantage of the zinc-binding protein, MAD data sets of zinc anomalous scattering were collected. In the future, combined homologous structure models and MAD phasing will be applied in the structure determination of D-Hydantoinase.

  

 

PS49

Structural Basis of the Lipid Binding in Rice Nonspecific Lipid Transfer Protein-1 Complexes from Oryza sativa

 

Hui-Chun Cheng*, Pei-Tsung Cheng, Peiyu Peng, Ping-Chiang Lyu and Yuh-Ju Sun*

Institute of Bioinformatics and Structural Biology, National Tsing Hua University,

Hsinchu 300, Taiwan, ROC

 

Nonspecific lipid transfer proteins (nsLTPs) facilitate the transfer of phospholipids, glycolipids, fatty acids, and steroids between membranes, with the binding affinity of a broad range.  Some nsLTPs exhibit anti-microbial activity in vitro.  Herein we report three crystal structures of rice nsLTP1 from Oryza sativa, complexed with myristic acid, palmitic acid and stearic acid.  This is the first rice nsLTP1 complex crystal structure has been reported.  One fatty acid binding site was observed in myristate and stearate complexes and two fatty acids binding sites were observed in palmitate complex.  The overall folding of rice nsLTP1 complexes belongs to the four-helix bundle with a long C-terminal segment, which is similar to other plant nsLTP1s.  The C-terminal loop region consists of the elasticity to accommodate various fatty acids and the number of molecules.  Meanwhile, the second lipid-binding site of rice nsLTP1 is depended on the amount of lipid molecules participating in the complex formation.  The most significant binding forces between the fatty acids and rice nsLTP1s proteins are the hydrophobic interactions from the binding cavity of proteins.  A significant conformational change of the C-terminal loop was found between the unliganded and liganded rice nsLTP1s. 

 

 

PS50

Folding and Chaperone Function of Escherichia coli Trigger Factor

Jun-Mei Zhou

National Laboratory of Macromolecules, Institute of Biophysics,

Chinese Academy of Sciences, Beijing 100101, China

 

Escherichia coli trigger factor (TF) is a multi-functional protein, it is involved in the maintenance of a translocation-competent conformation of the precursor protein proOmpA (outer membrane protein A), and is a peptidyl-prolyl cis-trans isomerase detected in the 50S subunit of functional ribosome. It is active in cooperating with chaperones such as GroEL and DnaK, and is the first chaperone met during nascent peptide folding in eubacteria. TF has a modular structure, containing three domains with distinct structural and functional properties. The guanidine hydrochloride (GdnHCl) induced folding of TF was investigated by monitoring Trp fluorescence, far-UV CD, second-derivative UV absorption, enzymatic activity and binding of the hydrophobic dye, 1-anilinonaphthalen-8-sulfonate (ANS). The native state of trigger factor was found to bind ANS in 1:1 stoichiometry with a Kd of 84 mM. A native like state, N’, show increased ANS binding, while retaining PPIase activity. N’ is stable around 0.5 M GdnHCl and is transiently populated during refolding. A compact denatured state, I, is populated around 1.0 M GdnHCl, is inactive and does not show significant binding to ANS. The data indicate that trigger factor unfolds in stepwise manner, consistent with its modular structure. The ability of TF to undergo structural rearrangement to increase hydrophobic binding while maintaining activity may be related to physiological function and provides a plausible mechanism for high affinity substrate binding.

 

 

PS51

Role of C-Terminal Peptide in Hepatitis E Virus Capsid Assembly

 

Joseph Wang,1,2* Li Xing,1 Tian-Cheng Li,3 Naokazu Takeda, 3 Tatsuo Miyamura, 3 Yasuhiro Yasutomi,

4 Darren J. Schofield,5 Suzanne U. Emerson, 5 Robert H. Purcell,5 and R Holland Cheng1

1 Department of Bioscience, Karolinska Institute, 14157 Huddinge, Sweden;

2 Institute of Public Health, National Yang-ming University, Taipei 112, Taiwan;

3Department of Virology II, National Institute of Infectious Disease, Shinjuku, Tokyo 162, Japan;

4 Department of Bioregulation, Mie University School of Medicine, Mie 514-8507, Japan;

5Hepatitis Viruses Section, Laboratory of Infectious Disease, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, Maryland 20852, Department of Bioscience at Novum,

Karolinska Institute , 14157 Huddinge, Sweden

 

The recombinant Hepatitis E virus-like particle (rHEV-VLP) is composed of 60 copies of structure protein ORF2 (pORF2) which, including amino acids 112-6081, form dimers dominate at icosahedral two-fold axis2. ORF2 protein has two domains, S domain forming the base shell and P domain forming the protruding spike. It was also found that pORF2 including residues 112-600 alone could not form capsid particles. Therefore the exclusion of the C-terminal residue 601 is crucial for the particles assembly. Here, we have attained a series of monoclonal antibodies that specifically recognize the C-terminal residues: HEP224 requires the epitope of residues 601-608 and HEP4 binds to the epitope of residue 578-6073. The 3D structures of rHEV-VLP complexed with antibody4, together with the 3D structure of chimeric rHEV-VLP in which B cell tag epitope of 11 amino acids were conjugated to pORF2 C-termini5, demonstrate that C-terminal region of pORF2 locates on the side of protruding dimmer, pointing towards the icosahedral five-fold axis. The C-terminal peptide orients as such that residue 608 locates at the top surface of pronounced domain while residue 601 is at the bottom region close to the shell domain. Our observation indicates that complete sequence to residue 601 may be essential for the proper folding of ORF2 protein and for dimer-dimer interaction. Furthermore, the C-termini of pORF2 may be free to the capsid assembly domains, as the rHEV-VLP forms without residues 609-660.

Reference: 1. Li, TC et al. (1997). J. Virol. 71:7207-7213. 2. Xing, L et al. (1999). Virology 265, 35-45. 3. Schofield, DJ et al. (2000). J. Virol. 74:5548-5555. 4. Cheng, R. H. (2000). Visualization on the grid of virus-host interactions. Lect Notes Comp Sci 13, 141-154. 5. Niikura, M et al. (2002). Virology 293, 273-280.

 

 

PS52

Structures of King Cobra Phospholipase A2

Sujuan Xu1, Hailong Zhang1, Lichuan Gu1, Yuyan Shu2, Shiying Song 1 and Zhengjiong Lin1*

1National Laboratory of Biomacromolecules, Institute of Biophysics,

Chinese Academy of Sciences, No.15 Datun Road, Beijing, 100101, China and

 2Institute of Snake Venom, Guangxi Medical University, No. 6 Binhu Road, Nanning, 530021, China

 

Phospholipase A2 (PLA2, EC 3.1.1.4) catalyzes the hydrolysis of the fatty acid ester at the sn-2 position of phospholipids with Ca2+ as an obligatory cofactor. Most PLA2s from snake venom possess a wide variety of pharmacological activities. The venom of Ophiophagus hannah  (king cobra) contains two homologous PLA2 enzymes. They belong to different subgroups, 1A and 1B. The enzyme belonging to subgroup 1B contains an unusual pancreatic loop (residues 62 to 66). This enzyme shows stronger toxicity (cardiotoxic and myotoxic activitiy) and lower PLA2 activity than the other homologous enzyme. Structures of these two PLA2s have been determined by X-ray crystallography. Significant conformational differences between the two PLA2s located at segment 15-19 and segment 58-67 around and including the pancreatic loop. These conformational differences may be related to the changes in toxicity and catalytic activity.

 

 

PS53

Multiple Conformations of Rubredoxin at 0.68 Å Ultrahigh Resolution

Reveal Its Electron Transfer Mechanism

 

Chun-Jung Chen 1*, Ming-Yih Liu2 , Yi-Ting Chen1 and Jean LeGall2

1Structural Biology Group, National Synchrotron Radiation Research Center, Hsinchu, Taiwan and

2Department of Biochemistry & Molecular Biology, University of Georgia, Athens, U.S.A.

No. 101, Hsin-Ann Road, Science-based Industrial Park, Hsinchu 30077, Taiwan

 

Rubredoxin (D.g. Rd) is a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas.  The protein is often purified from anaerobic bacteria where it is thought to be involved in electron transfer or exchange processes.  Rd transfers an electron to oxygen to form water as part of a unique electron transfer chain, composed by NADH:rubredoxin oxidoreductase (NRO), rubredoxin and rubredoxin:oxygen oxidoreductase (ROO) in D.g.  The crystal structure of D.g. Rd has been determined by Fe-SAD and direct method, and refined to ultra-high 0.68 Å resolution using synchrotron radiation X-ray.  Rd contains one iron atom bound in a tetrahedral coordination by the sulfur atoms of four cysteinyl residues. The hydrophobic and p-p interactions maintain the internal Rd folding. Multiple conformations of the iron-sulfur cluster and amino acid residues are observed and suggest it unique electron transfer mechanism. A number of hydrogen bonds, including N-H•••SG of the iron-sulfur are revealed clearly in electron density maps. This ultrahigh-resolution structure allows us to study the structure-and–function relationship of rubredoxin in great details, such as salt bridges, hydrogen bonds, water structures, cysteine ligands, iron-sulfur cluster, and electron and charge density distribution among activity sites. This will provide, for the first time, a clear role for this protein in a strict anaerobic bacterium.

 

 

PS54

Self-assembly of Vaccinia Viral Fusion Protein A27L Regulates Its Ability to Bind to Cell Surface Haparan Sulfates: Structural and Functional Approach via Site-specific Mutagenesis

 

Yung-Chang Peng 1*, Jye-Chian Hsiao 2, Che-Sheng Chung2, Ta-Hsien Lin 3.4, Wen Chang2 , and Der-Lii M. Tzou1

1Institute of Chemistry, Academia Sinica , 2 Institute of Molecular Biology, Academia Sinica,

3Department of Medical Research & Education, Taipei Veterans General Hospital,

 4Institute of Biochemistry, National Yang-Ming University

 

We have previously determined the structure of an extracellular domain of vaccinia viral envelope protein A27L that binds to cell surface heparan sulfate and mediates cell fusion during virus entry. Soluble A27L protein forms multimeric oligomers that, upon treatment with denaturing agent, retained residual α-helical content due to hydrophobic interactions among L47, L51 and L54. In this study, we analyzed the role of L47, L51 and L54 in biological activity by site-directed mutagenesis with leucine to alanine substitution to construct three single mutants, L47A, L51A and L54A and one triple mutant L47,51,54A in an attempt to disassociate the oligomeric structure.

All the mutants bind to heparin in vitro whilst the triple mutant does not bind to cell surface heparan sulfates nor does it block virus entry and cell fusion, indicating that the status of coiled-coils regulates the biological activity of A27L protein. Gel filtration, ciurcular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to probe the oligomeric stability of these A27L mutant proteins, respectively. We found that A27L protein forms stable hexamers that depend on close hydrophobic interactions among residues within the coiled-coil region.

The oligomeric states of single mutant were reduced to tetramer; however, the triple mutant became unstable and disassociated into monomer easily. In summary, our data indicate that hexameric oligomerization is critical for the biological role of A27L protein in virus entry and cell fusion.

 

  

PS55

NMR & Molecular Modeling Study the Conformations of the Inactivated Na+ Channel Gate Peptides Interacted with the Local Anesthetic Diphenyl Drugs

 

Bih-Show, Lou1*, Ta-Hsien, Lin2 and Chi-Zen, Lou2

1 Center of General Education, Chang Gung University, Tao-Yuan, Taiwan, ROC;

2Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan ROC

 

The local anesthetic effect is ascribable to the inhibition of Na+ current by selective binding of diphenyl drugs to the inactivated channels. Two important molecular determinates in the chemical structure of these drugs, namely two benzene rings separated by a center to center distance of ~5 Å and stem bond angle ~ 110°. Several diphenyl drugs such as phenytoin, carbamazepine, diphenhydramine, imipramine will be selected to interact with model peptides that their sequence corresponds to the linker part of rat brain type IIA Na+ channel. The NMR spectroscopy and molecular modeling will be applied in this study. The present of this work are to closely look at the detail orientation of diphenyl groups in desirable structures of selected drugs, and study the effect of structural and conformational properties related to inhibition of inactivated Na+ channel. Further molecular modeling will be performed to identify possible inhibitor-receptor interactions that may be critical for recognition and signal transduction of inactivated Na+ channel. NMR spectra of three diphenyl drugs (such as phenytoin, diphenhydramine, phenylbutazone) from three different drug groups in presence of two model peptides in both phosphate buffer and lipid (DMPC/DHPC), which micelles serve to mimic the peptide-lipid interactions, will be measured to obtain information on the interactions between three different drug groups.

 

 

PS56

Study on the vitamin E protection of lens proteins against lipid

peroxidation by FT-Raman spectroscopy

 

Shoeher Shih1,Yih-Ming Weng1, and Wenlung Chen2*

1Dept. of Food Science and 2Dept. of Applied Chemistry, National Chiayi University, Chiayi, Taiwan.

No.300 University Road, Chiayi, 60083, Taiwan

 

Vitamin E is the major antioxidant to the lipid peroxidation in biological membrane. How does it protect lens proteins from oxidative insults remain elucidated. We presented here the Raman spectral characterization of lens proteins of Tilapia lens protected by vitamin E against lipid peroxidation stress. It showed that water-soluble protein and urea-insoluble protein were relatively inert to the dietary vitamin E, while urea-soluble protein was affected sensitively to the dietary vitamin E. In addition, vitamin E also influenced the glutathione (GSH) level in Tilapia’s lens which might cooperatively protect lens proteins from lipid peroxidative attack.

 

 

PS57

Structural Comparison of Wild-Type and the C192S Mutant of

Streptococcal Pyrogenic Exotoxin B

 

 

Hsiang-Chee Houng*, Shih-Chi Lo, and Woei-Jer Chuang

Department of Biochemistry, National Cheng Kung University College of Medicine, Tainan 701, Taiwan,

No.1, University Road, Tainan, 701, Taiwan

 

Streptococcal pyrogenic exotoxin B (SPE B) is an extracellular cysteine protease produced by the pathogenic bacterium Streptococcus pyogene. SPE B is initially expressed as a 40-kDa zymogen, and subsequently converted to 28-kDa active protease by autocatalysis or proteolysis. The mature protease was shown to participate in the dissemination, colonization, and invasion of bacteria and the inhibition of wound healing. This suggests that SPE B serves as an important virulence factor in streptococcal infections, making it an attractive therapeutic target. In order to investigate the structure and function relationships of SPE B, we expressed SPE B and its inactive C192S mutant in E. coli and purified them to homogeneity. In this study, we determined the backbone 1H, 13C, and 15N resonances for the 28-kDa wild-type and the C192S mutant of SPE B and deduced their secondary structures from multidimensional NMR spectroscopy. The chemical shift perturbation analysis suggests that 28-kDa wild-type and the inactive C192S mutant of SPE B have the same tertiary fold. The major conformational differences are found in the residues C192, S282, H340, and W357.

 

 

PS58

Structure-Function Relationships of RGD-containing proteins in

Integrin αIIbβ3, αvβ3, and α5β1 Recognition

 

 

Yu-Chen Liu* , Jia-Hau Shiu , Chiu-Yueh Chen, and Woei-Jer Chuang

Department of Biochemistry, National Cheng Kung University, Tainan 701, Taiwan,

No.1, University Road, Tainan, 701, Taiwan

 

The RGD sequence is the cell attachment site of a large number of adhesive ECM, blood, and cell surface proteins, and nearly half of 24 known integrins recognize this sequence in their adhesive ligands. Rhodostomin (Rho) is an RGD-containing protein and a potent platelet aggregation inhibitor. Our previous report showed that Rho expressed in P. pastoris possesses the same function and structure as native protein. From the analyses of platelet aggregation and cell adhesion, we found that the mutant proteins containing the sequences AKGDWN and ARGDDL in the RGD loop can selectively inhibit integrins αIIbβ3 and αvβ3, respectively. In contrast, the mutant protein containing the sequences ARGDXP in the RGD loop have better inhibitory activity in integrin α5β1. To understand structural requirement for integrin recognition, we determined 3D structures of wild-type (PRGDMP) and mutant proteins (ARGDWN, AKGDWN, ARGDDL, ARGDMP, and ARGDNP) of Rho by using NMR spectroscopy. Based on our NMR analyses, the tertiary structures of these proteins are the same. The RGD conformations of these proteins are quite similar, expect that the residue D of ARGDDL protein has different orientation and the residue W of AKGDWN and ARGDWN interacts with the residue A and the C-terminus. These results indicate that the orientation of the D residue of the RGD motif and the C-terminal region of Rho play important roles in integrin recognition.

  

 

PS59

Expression in E. coli and NMR Characterization of Human Cytochrome c

Wen-Yih Jeng*, Yu-Hong Tsai and Woei-Jer Chuang

Department of Biochemistry, National Cheng Kung University Medical College, Tainan 701, Taiwan,

 No. 1, University Road, Tainan, 701, Taiwan, ROC

 

Cytochrome c is a heme protein involved in electron transfer, cell apoptosis, and diseases associated with oxidative stress. Here we synthesized the gene of human cytochrome c with E. coli favorable codons and then co-expressed the structural genes for human cytochrome c and yeast cytochrome c heme lyase in E. coli. We report a simple purification procedure to purify recombinant cytochrome c, and the yields of human holocytochrome c obtained from E. coli were 10-15 mg/L. The redox potential of recombinant human cytochrome c was 0.246 V which was measured by cyclic volatmmetry measurement. This is similar to that of horse cytochrome c with a value of 0.249 V. The sequential assignment and structural analysis of recombinant human ferrocytochrome c were obtained using multidimensional NMR spectroscopy. Structures were calculated by using the hybrid distance geometry-dynamical simulated annealing method. A total of 1477 NOEs, 33 hydrogen bonds, and 81 dihedral constraints were used for structure determination. The RMSD values with respect to the mean structure for the backbone and all heavy atoms of a family of 20 structures are 0.63 ± 0.10 and 1.06 ± 0.10, respectively. Based on our NMR studies, the recombinant human cytochrome c produced in E. coli exhibits the same tertiary fold as horse cytochrome c. These results provide the evidence that human cytochrome c expressed in E. coli possesses similar function and structure as those of horse protein. It is known that cytochrome c plays a role in many human diseases. This study serves as the basis for gaining insight into human diseases by exploring structure and function relationships of cytochrome c to its interacting proteins.

 

 

PS60

Solution Structure of the Domain III of the Japanese Encephalitis

Virus Envelope Protein

 

Ya-Ping Tsao*, Kuen-Phon Wu, Chih-Wei Wu and Chih-Wei Wu

Institute of Biotechnology and Department of Life Science,

National Tsing Hua University, Hsinchu 300, Taiwan.

101, Section 2 Kuang Fu Road, Hsinchu, Taiwan 300, Republic of China

 

Flaviviruses are small (50 nm) positive-strand RNA viruses that contain a lipid-bilayer membrane. The flavivirus envelope protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. We have determined the solution structure of the major antigenic domain (domain III) of the Japanese Encephalitis Virus (JEV) envelope protein. The JEV domain III forms a β-barrel type structure composed of six antiparallel β-strands resembling the immunoglobulin constant domain. In addition, we have identified the neutralizing epitopes of the JEV domain III to its monoclonal antibody (mAb E3.3) by comparing NMR chemical shifts of the free (14 kD) and the antibody-bound (178 kD) forms. Our results provide a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses.

  

 

PS61

The quaternary structural variance of human apolipoprotein E3 and E4:

Size distribution analysis by analytical ultracentrifugation

 

Chi-Yuan Chou1, Ming-Shi Shiao2and Gu-Gang Chang3

1Institute of Life Science, National Defense Medical Center, Neihu, Taipei 114, 2Department of

Medical Research and Education, Veterans General Hospital-Taipei, Taipei 112, and 3Faculty of

Life Sciences, National Yang-Ming Universities, Taipei 112

No.155 Li-Nong Street, Section 2, Shih-Pai, Taipei, 112, Taiwan

 

Human apolipoprotein E (apoE) is a 299 amino acid protein with molecular weight of 34 kDa. There are three main isoforms of apoE, designated E2, E3, and E4, coded by three common alleles, ε2, ε3, andε4 at a single gene locus on human chromosome 19q13.2. Their differences are single amino acid substitutions involving cysteine-arginine replacements at residue 112 and 158. ApoE3 (Cys112, Arg158) is the most common isoform whereas apoE4 (Arg112, Arg158) is associated with both familial late-onset and sporadic Alzheimer’s disease (AD). Accumulated apoE4 or its C-terminus is found in the senile plaques and neurofibrillary tangles seen in postmortem examinations of the brains of AD patients. AD patients who are homozygous for the e4 allele exhibit more highly developed senile plaques at autopsy.

In this study, we have expressed and purified the full length and N-terminal truncated E3 and E4 from Escherichia coli and compared their quaternary species distribution in phosphate buffer saline. By the experiments of sedimentation velocity and continuous size distribution analysis, the full-length E3 and E4 proteins showed a very similar distribution pattern in which there were 9-10 species with sedimentation coefficients (S) between 3.5 and 27.0. The N-terminal forty amino acids (aa) - truncated E3 showed a major species with S of 4.5 (64%) and the truncated E4 showed two major ones with S of 3.6 (26%) and 4.9 (51%). Furthermore, the N-terminal seventy-one aa - truncated E3 showed a major species with S of 5.9 (77%), and very differently, the same truncated E4 showed more complicate species distribution from S = 4.0 to 23.0. The variety in the quaternary species distribution of N-terminal truncated E3 and E4 give us some information to explain the more easily aggregating feature of E4 and the correlation of E4 and AD.

 

 

 

PS62

Anaerobic Structure of Ferredoxin II from Desulfovibrio gigas

 

Yin-Cheng Xie 1,3*, Ming-Yih Liu 2, Jean LeGall2, Chun-Jung Chen 1*

1 Structural Biology Group, National Synchrotron Radiation Research Center, Hsinchu, Taiwan

2 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, U.S.A.

3 Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu, Taiwan

 

Ferredoxin II (Fd II) is a small electron transfer protein, isolated from the strict anaerobic sulfate-reducing bacterium, Desulfovibrio gigas.  It contains 58 amino acids and a [3Fe-4S] cluster. Anaerobic Fd II crystals have been grown using hanging-drop vapor diffusion method under anaerobic condition. Fd II crystals diffracted to a resolution of 1.4Å and belong to space group P21212, with unit-cell parameters a= 32.685 Å, b= 82.281 Å, c= 22.953 Å.  The structure of anaerobic Fd II has been determined and refined. Its [3Fe-4S] cluster is bound with Cys8, Cys14 and Cys50, and Cys11 extends away from cluster. Cys18 and Cys42 form a disulfide bond to maintain foldings.  In addition to a [3Fe-4S] cluster, five isolated heavy electron densities around the anaerobic ferredoxin II are located, and the density heights are similar to those of cluster irons.  These extra irons are bound with Glu, Asn and Asp, respectively, which reveals the unique iron-storage function and electron transfer pathway of ferredoxin II. The structure comparison between aerobic and anaerobic Fd II is also discussed.

 

 

PS63

Binding Mechanism of Non-specific Lipid Transfer Proteins and their role

in Plant Defense

Chao Sheng Cheng, Dharmaraj Samuel, Yaw Jen Liu, Ku Feng Lin and Ping-Chiang Lyu*                      Department of Life Science, National Tsing Hua University, Taiwan

101, Section 2 Kuang Fu Road, Hsinchu, Taiwan 300, Republic of China

 

Plant non-specific Lipid Transfer Proteins (nsLTPs) are small basic proteins that can transport phospholipids between membranes.  Based on the molecular weight, nsLTPs are subdivided into nsLTP1 and nsLTP2.  NsLTPs are all helical proteins stabilized by four conserved disulfide bonds.  Existence of an internal hydrophobic cavity, which has been proven to be the binding site for lipid-like substrates, is a typical characteristic of nsLTPs.  NsLTPs are known to participate in the plant defense, but the exact mechanism of their antimicrobial action against fungi or bacteria is still unclear.  A receptor at the plant surface recognizes fungal protein (elicitin) complexed with ergosterol to trigger plant defense responses.  It was proposed nsLTPs, which share high structural similarities with elicitin, need to be associate with a hydrophobic ligand to trigger the plant defense.  In this paper, we proposed that the potential hydrophobic ligand is sterol molecule through analyzing various biophysical methods.  NsLTP2 could accommodate the planar sterol molecule, but nsLTP1 lodges only linear lipid molecules.  Although the cavity size of rice nsLTP2 is smaller than rice nsLTP1, it is flexible enough to accommodate the voluminous sterol molecule.  The dissociation constant for nsLTP2/cholesterol complex is approximately 71.21 mM by way of H/D exchange method using mass spectroscopic technique.  A comparison of NMR spectra of sterol/rice nsLTP2 complex and free forms revealed the residues involved in the binding.  Schematic models of nsLTP structures give interesting clues about the reason for differential binding modes.

 

 

PS64

The Effect of pH and Double Helix Association on Pyrimidine Motif Triplex FormationThermodynamic and Kinetic Studies

 

Si-han Chen1*, Min-tasir Wey2, Chia-ching Chang1, Lou-sing Kan2

1Department of Physics, National Dong Hwa University, Hualien, Taiwan.

2 Institute of Chemistry, Academia Sinica, Nan-Kang, Taipei 11529, Taiwan.

 Institute of Chemistry, No.128 Academia Road Section 2, Nan-Kang, Taipei, 115, Taiwan

 

The requirement of protonation on cytosine of homopyrimidine triplex-formation oligonuleotide (TFO) severely limits its use in an artificial regulation of gene expression at physiological pH. To this end, we have investigated the pH dependence (from 4.6 to 7.0) and the effect of double helix association, on pyrimidine motif triplex formation by using isothermal titration calorimetry as a function of pH at 20. The triplex formation between a 17-mer double-stranded homopurine-homopyrimidine (defined as W-C) and a 17-mer single-stranded oligonuleotide (defined as H) at pH 4.6 is driven by a small negative enthalpy change, which is -24 kcal/mol, and it reduce 10 times when the pH raises to 5.6. The result of melting experiment suggests that the characteristic of Hoogsteen basepairing is quite different from Watson-Crick basepairing. And, the thermodynamic and kinetic studies indicate that the Hoogsteen hydrogen bonds formed between TFO and the target duplex suffer more stringent condition in a nucleation step than Watson-Crick hydrogen bonds of duplex formation, and are severely limited by pH. Further discussion of triplex-formation mechanism is also included in this study. We conclude that the formation of triplex exhibits a mixed or multistep type, instead of a simple two-state model. Furthermore, we use W and C as titrants separately, in order to examine the effect of double helix association on the triplex formation. The binding constant for triplex formation with the effect of duplex association is much larger by a factor of 15 or more than that without being affected by duplex association, and this effect on triplex formation is approximately pH independent. This study may shed a light on therapeutic application of the antigene strategy in vivo.

 

 

  

PS65

Calcium Induced Protein Folding: NMR study on a Protozoan

Calcium-Binding Protein

Sulakshana Mukherjee and K.V.R. Chary

Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai

 400 005 India

 

One of the calcium-binding proteins from Entamoeba Histolytica (EhCaBP) is a 134 amino acid residue long EF-hand protein (Mr ~ 15 kDa). The three-dimensional (3D) structure of this protein in its holo form has been recently determined. However, the 3D structure of Ca2+ free (Apo) form of this protein is not yet known. In light of this, we have attempted to structurally characterize the apo-state of this protein. The outcome of such efforts is presented in this presentation. As a first step in this direction, we have carried out Ca2+ titration with the Apo-form of EhCaBP using NMR. Such titration reveals the folding of the N-terminal domain prior to the C-terminal domain. Further, the deuterium exchange studies threw light upon the structure of Apo-EhCaBP, which has some of the helices and all the b-strands intact, as seen in its holo form. However, the tertiary structure of the protein seen in its holo form, has collapsed which proves that the Apo-form of EhCaBP is a molten-globule.

 

 

PS66

The effect of metal ion in the maintenance of the structure

Integrity of malic enzyme

Sheree Lee

National Yang-Ming University

 

Malic enzyme exists widely in nature. It catalyzes the reversible oxidative decarboxylation of malate to pyruvate and CO2, with concomitant reduction of NAD+ or NADP+ to NADH or NADPH. In addition to NAD(P)+ , the enzyme also requires divalent metal ions (Mn2+ or Mg2+) as cofactor.

In the present report, we study the unfolding of pigeon malic enzyme in urea. The structure changes of the enzyme molecule was monitored with circular dichroism, fluorescence, and analytical ultracentrifuge. The results revealed biphasic conformational change in secondary and tertiary structure. There is an intermediate state at 3 ~ 5 M urea. In the presence of 4 mM Mn, the intermediate state disappeared and the urea denaturation pattern changed to monophasic. The signal changes of analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in lower urea concentration; In the intermediate state, the enzyme underwent polymerization. The polymers were dissociated to unfolded monomers at urea concentration greater than 6 M. Mn2+ retarded the polymerization of malic enzyme and could improve the conformational stability of malic enzyme. In thermostability experiment, metal ion had no protective effect; but metal ion could protect malic enzyme structure against digestion with trypsin.

D258 is a direct metal ligand site. We have replaced aspartic acid at 258 to asparagine. The mutant malic enzyme (D258N) with a defective metal ligand did not protect by Mn2+ any more. The quaternary structure change revealed that it polymerized in 3 ~ 5 M urea, then depolymerization (> 6 M) in the absence or presence of 4 mM Mn2+. Mn2+ could not stabilize the overall structure of D258N mutant in chemical denaturation or in trypsin digestion experiment.

In conclusion: the metal ion not only plays a catalytic role in malic enzyme, but also has protected effect by stabilizing the protein architecture.       

  

 

PS67

Crystal Structures of Nucleosie Diphosphate Kinase in Rice

Jen-Yen Huang1, Chia-Yu Chang2, Tschining Chang2 and Chun-Jung Chen 1

1Structural Biology Group, National Synchrotron Radiation Research Center, Hsinchu, Taiwan and

 2Instiute of Biological chemistry , Academic Sinica, Taipei 11529,Taiwan.

 

Nucleoside diphosphate kinase (NDK) is a ubiquitous enzyme found in all organisms  (eukaryotes and prokaryotes) and cell type.  It catalyses the transfer of the tpphosphoryl group from a nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP).  NDK is involved and required for coleoptile elongation in rice.  The level of the enzyme changes during seed germination and the early stages of seedling growth.  Although several NDK structures from bacteria and animals have previously been available, the structures from plants are not yet determined.  Crystals of the rice NDK have been obtained and several sets of data were collected at SPring-8 BL12B2 in Japan.  The crystals belong to the space group P2(1)2(1)2(1) with unit-cell parameters a = 70.98 Å, b = 182.26 Å, c = 188.30 Å, and diffract to 2.5 Å.  The rNDK structure shows the overall folding with four β-sheets surrounded by six α-helices.  The model contains 149 residues of a total 152 residues and averaged 18 water molecules.  Hexameric molecular packing in both crystals and solution implies the rice NDKs function as hexamers.  In this study, we have determined the first rice NDK structure, which provides the detailed structural-functional information to understand the functional significance of this enzyme during plants growth and development in rice as well as plants.

 

 

PS68

Structure, antibacterial and antitumor activities of three novel gadolinium complexes containing fluoroquinolones

Guoping Wang1,2 *, Qingsen Yu1, Shangwu Ding2

1Department of Chemistry, Zhejiang University, 38 Zheda Road, Hangzhou, 310027, Zhejiang, China;

2Department of Chemistry, National Sun Yat-sen University, 70 Lien-Hai Road,

Kaohsiung 80424, Taiwan, ROC.

 

Fluoroquinolones and their metal complexes are the widely used antibiotics for treating numerous diseases. The synthesis procedures of these compounds have been well established and their physicochemical properties characterized. Their antibacterial properties were also tested. There have been very few publications on the antitumor properties of quinolones metal complexes. Recently, three novel Gadolinium complexes of fluoroquinolone, Gd (L)3·6H2O {L= Norfloxacin (NFLX), Ofoxacin (OFLX) and Ciprofloxacin (CPLX), respectively}, have been synthesized by us. The compounds have been characterized with FT-IR, molar conductivity, elemental analyses, DTA, TG, and electron paramagnetic resonance (EPR). In all of the complexes, the ligand L is coordinated through Ocarbox and Oketo atoms. Both ligands and complexes are assayed against gram-positive and gram-negative bacteria by in vitro doubling dilutions method, the complexes show the same minimal inhibitory concentration (MIC) as the corresponding ligands against Staphylococcus Aureus, Micrococcus Lutens, Escherichia Coli and Pseudomonas Aeruginosa. The inhibitory effect of the ligands and complexes on leukemia HL-60 cell line has been measured by using MTT (Methyl-Thiazol- Tetrozolium) assay method and liver cancer BEL-7402 cell line measured by SRB (Sulphurhodamin B) method. The results indicate that the complex Gd(OFLX)3·6H2O has strong inhibitory effect on BEL-7402 cell line and Gd(CPLX)3·6H2O has strong inhibitory effect on HL-60 and BEL-7402 cell lines. The relationship between structure and activities and the proposed antitumor mechanism of these compounds has been explored.

 

 


 

PS69

A novel analysis approach to investigating protein dynamics with nuclear magnetic resonance spectroscopy

Li-Chun Huang*1, Ren-Ting Wang2, Mei-Hui Guo2, Shangwu Ding1

1Department of Chemistry, National Sun Yat-Sen University,

70 Lien-Hai Road, Kaohsiung, Taiwan 80424, Republic of China;

2Department of Applied Mathematics, National Sun Yat-Sen University,

70 Lien-Hai Road, Kaohsiung, Taiwan 80424, Republic of China.

 

NMR spectroscopy provides unique information on dynamics of biological macromolecules. For proteins, several methods have been proposed to yield order parameters of backbone N-H vector and side-chain, fast and slow (effective) correlation times etc. These methods differ from each other mostly by using different models for motions in proteins. These methods either use simplistic analytical solutions, or perform linear regression, to extract dynamic parameters from relaxation rates. This may bring unusually large error, particularly for loops and residues with labile protons. It also limits the number of unknowns in the analysis. A novel approach based on non-linear regression analysis of the relaxation rates is proposed. In addition to permitting more unknowns in data analysis, this method offers better precision in determining dynamical parameters (order parameter, correlation times etc.). These features have been verified with three real and typical proteins (human ubiqitin, α2D and βARK1 PH domain). The potential of this approach applied to more versatile systems such as membrane proteins is explored.

 

 

PS70

Revisit to dissolution of amino acids in water with liquid state NMR spectroscopy

Hui-Jun Wang, Shangwu Ding*

Department of Chemistry, National Sun Yat-Sen University,

70 Lien-Hai Road, Kaohsiung, Taiwan 80424, Republic of China

 

Detailed description of solvation dynamics of biological molecules in water or other solvents has been regarded as one of the most important problems in biological physics. Recently much attention has been focused on the understanding of protein-solvent interaction and its significance to physiological functions and biological evolution of organisms. Although the most relevant objective of research in this area is proteins, the study of amino acids is enlightening and benefiting. With high resolution NMR spectroscopy at different magnetic fields, we have performed a detailed investigation of the solvation dynamics of amino acids in water. We have obtained systematical experimental results of the interaction between amino acids and water. Some important findings have been made. For instances, the presence of surface water, typical of longer correlation times compared to bulk water, has been established; the transitional layers of water between surface and bulk water have also been observed. The differences of solvation dynamics among hydrophilic, hydrophobia and charged amino acids are shown and explained. The implication of these results to more general aspects of the interaction between biological molecules and solvents is discussed.

 

 


 

PS71

Applications of MQMAS spin-diffusion spectroscopy of half-integer quadrupolar spins to biological solids

Shun-Liang Tseng1, Zhihong Liu2, Jian-Ming Chen1, Huan-Sheng Huang1, Kunta Li1,

Ming-Yuan Liao3, Shangwu Ding1*

1Department of Chemistry, National Sun Yat-Sen University,70 Lien-Hai Road, Kaohsiung,

 Taiwan 80424, Republic of China;

2Wuhan Institute of Physics and Mathematics, The Chinese Academy of Sciences, Wuhan 430071, China;

3Department of Applied Chemistry, National Chi-Nan University,

1 University Road, Puli, Taiwan 403 , Republic of China.

 

The lineshape of the cross peaks in a spin diffusion or exchange spectrum of a half-integer quadrupolar spin systems, such as Na-23, Al-27, Zn-67, contain the information of the relative orientation of the electric-field-gradient (EFG) tensors of a pair of coupled such spins. This information is extremely useful in determining the electric and geometric structure as well as dynamics of a variety of compounds and materials. For half-integer quadrupolar spin systems, this information has been elusive due to insufficient resolution caused by second and high orders of quadrupolar broadening. The invention of mutli-quantum magic-angle spinning (MQMAS) technique greatly alleviates the resolution problem and spectral spin diffusion spectra with acceptable resolution of half-integer quadrupolar spin systems can be achieved. Furthermore, this lab proposed the application of recoupling techniques to improving spin diffusion efficiency. With these progresses, MQMAS has become a powerful tool for studying the structural and dynamical characteristics of biological systems. In this work, we will report the above progress and the results of applying this improved method to a number of biologically interested systems such as ATP sodium salts and metalloproteins.

 

 

PS72

Calcium-Dependent Protein-Protein Interactions Induce Changes in Proximity Relationships of Cys-48 and Cys-64 in Chicken Skeletal Troponin I

Y.M Liou *. Ming-Wei Chen.

Department of Life Science, Institute of Biochemistry, National Chung-Hsing University, Taichung, 402, Taiwan

250 Kuokang Road, Department of Life Science, National Chung-Hsing University

 

This study was to determine proximity relationships between two cysteine residues (Cys-48 and -64) in the N-terminal domain of chicken skeletal TnI associated with the Ca2+-dependent protein-protein interactions for muscle contraction. These two residues were labeled with two sulfhydryl-reactive pyrene-containing fluorophores (N-(1-pyrene)maleimide, and N-(1-pyrene)iodoacetamide). The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence (380~ 400 nm) and a broad peak of excimer fluorescence (480~490 nm). The excimer fluorescence arises from the tendency of two adjacent pyrene groups to form a dimer, and the magnitude of the excimer fluorescence indicates the ability of the two pyrene groups to approach to within about 5 Å of each other. Results obtained show that Cys-48 and Cys-64 are sufficiently close to each other to give rises to an excimer peak. Forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca2+ decreases the excimer fluorescence and separates these two residues. This reduction in excimer fluorescence does not occur in the labeled TnI complex with cardiac TnC (cTnC). When Ca2+ binds to the two N-terminal sites of sTnC, a further decrease in excimer fluorescence and an increase in monomer fluorescence were found in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus, the N-terminal region of TnI may participate in the Ca 2+-activation of muscle contraction.

  

 

PS73

A Non-Native a-Helix Is Formed in the b-Sheet Region of the Molten Globule State of Canine Milk Lysozyme

Masahiro Watanabe1*, Yoshihiro Kobashigawa1,2, Tomoyasu Aizawa1, Makoto Demura1, and Katsutoshi Nitta1

1Division of Biological Sciences, Graduate School of Science, Hokkaido University and 2Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST)

1Kita-10 Nishi-8 Kita-ku, Sapporo city, 060-0810, Japan and 22-1 Tsukisamu Higashi Toyohira-ku,

Sapporo city, 062-8517, Japan

 

The native and the molten globule states (N and MG states, respectively) of canine milk lysozyme (CML) were examined by CD spectroscopy. The absolute ellipticity value at 222 nm of the MG state was larger than that of the N-state, indicating that the helical content of the MG state was higher than that of the N-state. It suggests that non-native a-helix is formed in the MG state of CML. We used the AGADIR algorithm, a helix-coil prediction program, to observe the helical propensity in the region in which the third b-strand was formed in the N-state. The results indicated the possibility of a-helix formation in the third b-strand region in the MG state of CML. To investigate this possibility, we designed a mutant, Q58P CML, in which the helical propensity of the MG state was significantly decreased around the third b-strand region. It appeared that the absolute ellipticity value at 222 nm of the CML mutant in the MG state was smaller than that of the wild-type protein. It could be assumed that the non-native a-helix is formed around the third  b-strand region of wild-type CML in the MG state. In addition, a guanidine-induced unfolding experiment revealed that the MG state of the Q58P CML mutant possesses thermodynamic stability and cooperativity of unfolding reaction almost identical to those of the wild-type protein, indicating that the non-native a-helix around the third b-strand region is formed by non-cooperative interaction between neighboring amino acid residues.

 

 

PS74

SDS Surfactant Influences on the Solution Structures of bata-Amyloid

peptide 1-40

Tsang-Lang Lin1*, U-Ser Jeng2 , Yuan Hu1, Zyun-Hua Huang1, and Derek L. Ho3

1 Department of Engineering and System Science, National Tsing Hua University, Hsinchu 30043, Taiwan.

Address. 101, Kuan-Fu Rd. HsinChu, 30043, Taiwan

2 National Synchrotron Radiation Research Center.  Address. 101 Hsin-Ann Road, Science-Based Industrial Park,

Hsinchu 30077, Taiwan, R. O. C.

3 National Institute of Standards and Technology, Gaithersburg, MD 02899, USA

 

In solutions, the major protein constituent of amyloid peptide, beta-amyloid peptide, folds into a random coil, beta-helix, or oligiemeric beta-sheet structure. Without buffering, beta-amyloid peptides evolve into the beta-sheet structure, and further form fibril that can deposit in Alzheimer disease. Here, we study the structural suppression effect of a surfactant, sodium dodecyl sulfate (SDS), on the formation of beta-sheet as well as fibril structures of beta-amyloid. Using circular dichroism (CD) for the secondary structure, we have observed that SDS can effectively suppress the formation of the beta-sheet structure, and maintain the peptides (0.1 mM) mainly in the beta-helix structure in water solutions. With small angle neutron scattering (SANS), we have also found the formation of peptide-surfactant complex of these beta-helix peptides with SDS. With a selected deuteration on SDS for contrast variation in SANS, we have extracted a detailed structural information of the peptide/surfactant complex, including the short rod-like shape, size, and the association ratio between the SDS and the peptide.

 

  

PS75

Thermodynamics and Kinetics of Protein Folding in the Mean Field Approximation

Ying-Jen Shiu1*, Charlene Su1, Kuo Kan Liang1 , M. Hayashi2 and S. H. Lin1,3

1Institute of atomic and molecular Sciences, Academia Sinica, Taipei, Taiwan,

2Center for Condensed Matter Sciences, National Taiwan University, Taipei, Taiwan and

 3Department of Chemistry National Taiwan University, Taipei, Taiwan

P.O. Box 23-166, Taipei, Taiwan

 

The kinetic Ising model in the mean field approximation has been applied to study the equilibrium and kinetic behaviors of protein folding-unfolding. Thermodynamics and kinetics of protein folding-unfolding are related by and elementary process of folding  unfolding of peptide bonds. We shall now show that even for the so-called two-state case of protein folding-unfolding, the kinetic behaviors are predicted to be in general non-exponential and universal plots exist separately for the thermodynamics behaviors and kinetics behaviors of protein folding-unfolding. Some experiment results have been analyzed by using this model.

 

 

PS76

Elucidation of the three dimensional structure of the chromo-3 sub-domain

of the Chloroplast Signal Recognition Particle

 

P. S. T. Leena, T. K. S. Kumar and Chin Yu

Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan. R.O.C.

 

The Signal recognition particle (SRP) plays a vital role in co-translational protein targeting across membranes.  The SRP present in chloroplasts (Cp SRP) consists of two domains, a 54 KDa (SRP54) domain and a 43 KDa domain (SRP43). The CpSRP is involved in the targeting of the light harvesting chlorophyll binding protein (LHCP) to the thylakoids.  The LHCP recognition by CpSRP is proposed to depend on the SRP43.  Four distinct structural sub-domains (Chromo1, Chromo2, Ank(1-4) and Chromo3) exist in SRP43. Although the exact functional role of the various SRP43 sub-domains has still not been delineated, the Chromo 3 sub-domain is believed to be involved in the downstream targeting of the SRP-LHCP complex.  The goal of the present study is to elucidate the three-dimensional structure of the Chromo 3 sub-domain using a variety of multidimensional NMR techniques.  Chromo3 sub-domain is expressed as a GST-fusion protein in E.coli in high yields (mg/l).  The protein is purified to homogeneity by GST-Sepharose affinity chromatography.  Far UV circular dichroism spectrum reveals the presence of helical segments in the protein.  In addition equilibrium urea-induced unfolding experiments showed that the chromo3 sub-domain is maximally stable at around neutral pH.  Partial resonance (1H, 15N & 13C) assignment of Chromo3 sub-domain has been accomplished based on the information content in the two and three-dimensional NMR spectra.  These results would be discussed in detail.

 

  

PS77

Sheared-type Ganti·Csyn Base Pair: A Unique d(GXC)  Loop Closure Motif

Ko-Hsin Chin1 & Shan-Ho Chou2*

1Institute of Biochemistry, National Chung-Hsing University, Taichung, 40227, Taiwan

2 Department of Life Science, National Central University, Jung-Li, 320, Taiwan

Institute of Biochemistry, National Chung-Hsing University, Taichung, 40227, Taiwan

 

Stable DNA loop structures closed by a novel G·C base pair have been determined for the single-residue d(GXC) loops (X = A, T, G, C) in low salt solution by high resolution nuclear magnetic resonance (NMR) techniques. The closing G·C base pair in these loops is not of the canonical Watson-Crick type, but adopts instead a unique sheared-type (trans Watson-Crick/Sugar-edge) pairing as those occurring in the sheared mismatched G·A or A·C base pair to draw near the two opposite strands. The cytidine residue in the closing base pair is transformed into the rare syn domain to form two H-bonds with the guanine base and to prevent the steric clash between the G2NH2 and the CH5 protons. Besides, the sugar pucker of the syn cytidine is still located in the regular C2’-endo domain, unlike the C3’-endo domain adopted for the pyrimidines of the out-of-alternation left-handed Z-DNA structure. The facile formation of the compact d(GXC) loops closed by a unique sheared-type Ganti·Csyn base pair demonstrates the great potential of the single-stranded d(GXC) triplet repeats to fold into stable hairpins.