Structural basis of membrane-induced cardiotoxin A3 oligomerization

Chwan-Deng Hsiao1Farhad Forouhar1, Wei-Ning Huang2, Jyung-Hurng Liu1 , Kun-Yi Chien2, and Wen-guey Wu2

1Institute of Molecular Biology, Academia Sinica, Taipe 115, Taiwan and

 2Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, 300 Taiwan


Cobra cardiotoxins (CTXs) have previously been shown to induce membrane fusion of vesicles formed by phospholipids such as cardiolipin or sphingomyelin.  It can also form pore in membrane bilayers containing anionic lipid such as phsphatidylserine or phosphatidylglycerol.  Herein, we show that the interaction of CTX with negatively-charged lipids causes CTX dimerization, an important intermediate for the eventual oligomerization of CTX during the CTX-induced fusion and pore formation process.  The structural basis of the lipid-induced oligomerization of CTX A3, a major CTX from Naja atra, is then illustrated by the crystal structure of CTX A3 in complex with sodium dodecyl sulfate (SDS), likely mimics anionic lipids of the membrane, under micelle condition at 1.9 Å resolution.  The crystal packing reveals a distinct SDS free and SDS rich regions, in the latter of which two types of interconnecting CTX A3 dimers, D1 and D2, and several SDS molecules can be identified to stabilize D1 and D2 by simultaneously interacting with residues at each dimer interface.  When the three CTX-SDS complexes in the asymmetric unit are overlaid, the orientation of CTX A3 monomers relative to the SDS molecules in the crystal is strikingly similar to that of the toxin with respect to model membranes as determined by NMR and FTIR methods.  These results not only illustrate how lipid-induced CTX dimer formation may be transformed into oligomers either as inverted micelles of fusion intermediates or as membrane pore of anionic lipid bilayers, but also underscore a potential role for SDS in X-ray diffraction study of protein-membrane interactions in the future.




The Proteolytic Analysis of the pMOB subunit from the Particulate Methane Monooxygenase (pMMO) 


Shan-Jen Kuo, Steve S.-F. Yu, Yu-Ju Chen, Hsin-Kai Liao, Sunney I. Chan

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

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


The Proteolytic Analysis of the pMOB subunit from the Particulate Methano Monooxygenase (pMMO) Mehanotrophic bacteria use methane as their feedstock and energy source. Particulate methane monooxygenase (pMMO), one of the membrane proteins converting methane to methanol, are enormous expressed (90% in membrane proteins) in these bacteria under high copper concentrations. pMMO contains three subunits: a-, b-, g-subunits.  Those subunits have been characterized through N-terminal sequence method and identified as 27 kDa, 45 kDa and 23 kDa, respectively. pMMO is less well studied than sMMO (soluble methane monooxyenase) due to the difficulty to obtain the well-resolved optical spectroscopic data in the presence of the membranes as well as the instability upon removal of the proteins from the membrane lipids. To overcome this circumstance, we, herein, developed a method to resolve the structural in formation directly when the proteins are still associated with the membrane. Proteins enriched membranes are easy to be separated from the soluble proteins through high-speed ultra-centrifugation operation.  Due to the hydrophobicity of the oil layer of the cell membrane, trypsin (cleavage site occurred at Lys and Arg) would be difficult to digest the membrane domains whereas the peptides located at the cytosolic exposed domains would be observed in the supernatants and could be characterized through the mass spectroscopic methods.  The appearances of the peptides fragments were studies will facilitate the insight of structural information regarding to the cytoplasmic domains and transmembrane domains.  To understand the three-dimensional fold and orientation of the secondary structure will provide us the detail about functional mechanism of the membrane proteins.




Phospholipid Membrane Interaction and Fusion Activity of Two Glutamate-rich Analogues of Influenza Hemagglutinin Fusion Peptide

Eric Assen B. Kantchev*, Shu-fang Cheng, Hung-jen Huang and Ding-kwo Chang

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

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


Membrane fusion, a ubiquitous cellular process, is directed in vivo by specialized membrane proteins. Influenza virus Hemagglutinin (HA) is the best-studied example of viral fusion proteins. It is cleaved by host proteases on two subunits, HA1 and HA2, linked by disulfide bonds. HA1 is involved in binding of the virion to receptors on the target cell. After endocytosis, HA2 triggers membrane fusion upon lowering of the pH inside the endosome. A highly conserved, hydrophobic stretch of 20 aminoacids at the N-terminus of the HA2 has been shown to play crucial role in the membrane fusion and has been named “fusion peptide” (FP). Wild type FP and single or multiple aminoacid mutants have been studied with a variety of biophysical methods (NMR, fluorescence, CD, IR). It has been shown that FP inserts in the outer leaflet of phospholipid bilayer at an oblique angle in the form of amphiphatic a-helix. We prepared a 25-mer synthetic FP (NH2-GLFGAIAGFIENGWEGMIDGWYGFR), and two glutamate-rich analogs, at residues 4,8 and 19 (E5(4,8)) and residues 3,7 and 19 (E5(3,7)) and labeled them with the fluorescent chromophores tetramethylrhodamine (Rho) and 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). CD-spectra in the presence of phospholipid (DMPC-DMPG = 1:1) vesicles showed that all peptides adopt predominantly a-helical form. From fluorescence experiments using Tryptophan or NBD we deduced that the peptides partition in phospholipd bilayer at both neutral and acidic pH, while inserting deeper at acidic pH. Self-quenching of Rho was used to probe the aggregation propensity of these peptides at low and neutral pH in the presence of phospholipid vesicles. Finally, the fusion activity of the E5 analogs was determined using FRET-based lipid mixing essay. We observed that both E5 analogs have similar properties to the wild type. Lipid binding, insertion depth and aggregation are diminished in the E5 analogs compared to the wild type, but the pH-dependence of these properties is enhanced. Furthermore, the effect of Glu substitution was more pronounced in E5(3,7) than E5(4,8). E5(3,7) analog was found to be approx. 10 times more fusogenic than the wild type.




An investigation on a cochlea neurotransmitter—

glutamate in acoustic trauma

Wenyuan Gao, Shengli Zhang, Lan Zhang, Haiying Liu, Mingfang Diao, Yinming Zhang

Department of Physiology, Zhang Hai Hospital, Shanghai 200433, China

No. 800 Xiang Yin Road, Department of Physiology, Shanghai 200433, China


The mechanism for bioenergetics and glutamate (Glu) excitotoxicity in acoustic trauma was explored. Guinea pigs were exposed to white noise at 115 dB SPL and content of glutamate was measured in perilymph by HPLC. The concentration of Glu significantly elevated after exposure. Agonists of Glu receptors NMDA and AMPA, antagonist MK-801, and an inhibitor of secretion of Glu, adenosine were then infused into cochlea respectively. NMDA and AMPA reduced the amplitude of CAP and elevated its threshold but had no effect on CM.  MK-801 and adenosine decreased threshold shift significantly after exposure. Nitric oxide (NO) and total antioxidant capacity (TAC) in cochlea changed obviously indicating that the production of reactive oxygen species (ROS) was connected with Glu in acoustic trauma. Administration of α-lipoic acid, a free radical scavenger, was found to protect hearing significantly in acoustic trauma. Conclusion: Glu, a cochlea neurotransmitter, plays an important role in acoustic trauma. Glu receptors NMDA and AMPA are involved in excitotoxicity. ROS is one of the primary mechanisms by which hearing is damaged.




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.




Permeation of Organic Ions through Calcium Channel in

Sarcoplasmic Reticulum Vesicles


Michiki Kasai*, Shinya Kobayashi, Hiroyuki Tsukagoshi and Yutaka Kanekawa

Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University

1-5 Yokotani, Seta Oe-cho, Otsu, Shiga 520-2194, Japan


Redox states of ryanodine receptor calcium release channel are important to regulate calcium release in sarcoplasmic reticulum (1).  It was suggested that the redox states are regulated by glutathione (1).  Recently, it was suggested that glutathiones of reduced state (GSH) and oxidized state (GSSG) permeated through ryanodine receptor calcium release channels in sarcoplasmic reticulum vesicles by using light scattering method (2). In that paper, they used fluorescence spectrometer to follow the light scattering intensity change. Since that result is very interesting, we followed that experiment by light scattering method using stopped flow apparatus to get more precise result.  We used sarcoplasmic reticulum vesicles prepared from rabbit skeletal muscle. The result showed that organic cations such as choline, lysine and arginine permeates through calcium channel and neutral molecules such as glucose and glysine do, because permeation of these molecules are dependent on calcium concentrations and enhanced by caffeine and ATP and blocked by ruthenium red and magnesium.  However, permeation of GSH was similar to that of organic anions such as glutamic acid and aspartic acid. Permeation of GSH was not affected by effectors for ryanodine receptor, but inhibited by DIDS and SITS which are known as inhibitors of anion permeation in sarcoplasmic reticulum (3). These results suggested that GSH or GSSG may permeate through anion channels existing in sarcoplasmic reticulum.

1. T. Oba, T. Murayama, and Y. Ogawa. Am. J. Physiol. Cell Physiol. 282, C684 (2002)

2. M. Csala et al. Biochem. Biophys. Res. Commun. 287, 696 (2001)

3. M. Kasai and T. Taguchi. Bioochim. Biophys. Acta 643, 213 (1981)




Conformational Change of Horseshoe Crab Antimicrobial Peptide,

Tachyplesin I, in Model Membrane


Mineyuki Mizuguchi1, Shinichi Kamata1, Shun-ichiro Kawabata2 and Keiichi Kawano1*

1Department of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2Department of Biology, Graduate School of Sciences, Kyushu University

2630 Sugitani, Toyama 930-0194, Japan


Tachyplesin I is a unique antimicrobial peptide in hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus.  Tachyplesin I inhibits growth of both Gram-negative and -positive bacteria at low concentration .  Furthermore, this peptide has antiviral activity against vesicular stomatitis virus, influenza A virus and human immunodeficiency virus (HIV)-I.  Tachyplesin I consists of 17 amino acid residues with two disulfide-bonds.  We determined the secondary structure of tachyplesin I using nuclear magnetic resonance (NMR) spectroscopy and showed that this peptide adopts an antiparallel b-sheet structure with slight bend connected by a b-turn (J. Biol. Chem., 265, 15365 (1990)).  It has been suggested that tachyplesin I interacts with lipid bilayer and destabilizes the membrane structure, concomitantly reducing cell viability.  We determined the solution structure of tachyplesin I in the presence and absence of artificial membrane phospholipid, dodecylphosphocholine (DPC) using two-dimensional NMR spectroscopy.  The bend in the antiparallel b-sheet is not seen in the structure with DPC and tachyplesin I adopts an extended b-sheet structure.  In this study, we will discuss the relationship between conformational changes induced by DPC and the mechanism of antimicrobial activity of tachyplesin I.




Microscopic Imaging of Membrane Physical Properties and Its

Applications to Liposomes and Cells


Taku Tanaka, Yukinori Taniguchi, Seigo Tsuchiya, Tetsuhiko Ohba and Kazuo Ohki*

Ddpartment of Physics, Graduate School of Science, Tohoku University,Aramaki

Aza-Aoba, Aoba-ku, Sendai 980-0861, Japan


The fluorescent probe, laurdan, changes its emission peak from 440nm in gel phase to 490nm in liquid-crystalline phase of lipid bilayer membranes. Using this unique property, we have constructed a video-rate microscopic imaging system, which can observe physical properties of membranes. The system is a fluorescence microscope equipped with home-build dual-view optics, image-intensified CCD camera and digital video recorder. The recorded images are processced to the image of generalized polarization (GP) defined by Parasassi et al. (Biophys. J. 57 (1990) 1179-1186). The GP image is a measure of membrane fluidity at video-rate.  We tried to remove the factors lowering performance such as chromatic aberration of an objective lens, polarization dependence of dichroic mirrors in the dual-view optics and slight distortion of image induced by the image-intensified optics in the camera, and we succeeded in obtaining the spatial resolution of GP images comparable with diffraction limit of optical microscopy. Using this instrument, we observed a clear phase separation in the slow cooling process of a giant liposome composed of DMPC and DMPE. And this phase separation was coupled with the shape change of liposome from round shape to angular one.  The imaging system was also applied to the study of apoptosis. When PC12D cells were grown in the medium containing 0.5 mM of hydrogen peroxide (H2O2) at 37 , the characteristic phenomena of apoptosis were observed at different times. The phosphatidylserine was exposed on the surface for 2h incubation.  The activity of cysteinylaspartate protease 3 was raised for 6 h incubation. The degradation of chromatin into small, membrane-wrapped fragments was observed for 12 h incubation. The membrane fluidity observed by the imaging system decreased for 0.5 h incubation and the change of membrane fluidity proceed the other apoptosis phenomena. These results imply that decrease of membrane fluidity on plasma membrane induced by H2O2 treatment triggers the initiation of apoptosis.




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.




Determination of the Recognition Sequences of Integrins aIIbb3, avb3, and a5b1 by Rhodostomin and Its Mutants

Chiu-Yueh Chen, Jia-Hau Shiu, Yi-Chun Chen, Yu-Chen Liu, and Woei-Jer Chaung.

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

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


Rhodostomin (Rho) is a potent inhibitor of platelet aggregation and consists of 68 amino acids including six disulfide bonds and a PRGDMP sequence at the positions of 48-53. Our previous report showed that Rho expressed in P. pastorispossesses the same function and structure as native protein. In order to identify the amino acid residues required for selective recognition of integrins aIIbb3, avb3, and a5b1, we mutate the residues in the RGD loop or C-terminal region of Rho, express the proteins in P. pastoris., and use the platelet aggregation and cell adhesion assays for inhibitory studies. We found that the mutant proteins containing the sequences AKGDWN (P48A/R49K/M52W/P53N) and ARGDDL(P48A /M52D/P53L) in the RGD loop can selectively inhibit Integrins aIIbb3,and avb3, respectively. The mutant proteins containing the sequences ARGDXP in the RGD loop have better inhibitory activity in integrin a5b1. These results suggest that the residues in or adjacent to the RGD loop of Rho play an important role in recognizing various integrins. In contrast, the C-terminal region has little effect on the interaction between Rho and aIIbb3 integrin.




Determination of the Recognition Sequences of Integrin a4b1

by Rhodostomin Mutants

Yi-Chun Chen*, Chiu-Yueh Chen, Jia-Hau Shiu, Yu-Chen Liu, and Woei-Jer Chaung.

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

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


Integrins are member of the large family of cell surface receptors that mediate cell adhesion, cell migration and signal transduction. Integrin a4b1 is an adhesive receptor that recognizes ILDVP sequence of the CS1 region of fibronectin and EILDV sequence of vascular cellular adhesion molecule 1 (VCAM-1). It is present on lymphocytes, monocytes, eosinophils, NK-cells, and thymocytes. Rhodostomin (Rho) is a potent inhibitor of platelet aggregation and consists of 68 amino acids including six disulfide bonds and a PRGDMP sequence at the positions of 48-53. Our previous report showed that Rho expressed in P. pastoris possesses the same function and structure as native protein. In order to identify the amino acid residues required for selective recognition of integrin a4b1, we mutate the residues in the RGD loop, express the proteins in Pichia p., and use the platelet aggregation and cell adhesion assays for inhibitory studies. We found that the mutant proteins containing the sequences EILDV (P48E/R49I/G50L/M52V) and IDSP (G50I/M52S) in the RGD loop cannot inhibit the platelet aggregation. Studies on the activity of Rho mutants in inhibiting integrin a4b1 to fibronectin or VCAM-1 are ongoing.




Identification of the residues of cytochrome c involved in

the interaction with Apaf-1


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

Department of Biochemistry, National Cheng Kung University College of Medicine,

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


Apoptosis is the dominant form of programmed cell death during embryonic development and normal tissue turnover. Cytochrome c can trigger the formation of the apoptosome that is a heptameric complex comprised of Apaf-1 (Apoptotic protease activating factor-1) and cytochrome c. The apoptosome binds and activates procaspase-9, resulting in activation of further caspases, such as caspase-3, which orchestrate the final packaging of the apoptotic cell. However, little is known about the interactions between cytochrome c and Apaf-1. Our previous report showed that human cytochrome c expressed in E. coli possesses the same function and structure as native protein. Based on the analyses of our human cytochrome c structure, we hypothesized that the residues K22, R38, K39, K53, K55, K72, K73, K86, and K87 of cytochrome c maybe involved in the interaction with the D residues of two WD-40 domains of Apaf-1. Therefore, we mutated these residues into alanine and expressed them in E. coli to test our hypothesis. The study on the microinjections of human cytochrome c and mutant proteins in the 293T and HeLa cells is ongoing.




Expression and Characterization of the DNA-Binding Domain of

FOXP1 Transcription Factor


Yuan-Ping Chu*, Chien-Hui Sun, I-Ju Yeh, Pei-Phen Liu and Woei-Jer Chaung

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

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


Forkhead box P1 (FOXP1) is a forkhead transcription factor, whichconsists of 677 amino acids with 3 isoforms and represses gene transcription via binding to the consensus site, TATTTRT. Analysis of the protein sequence of FOXP1 revealed that domains unique to FOXP1 included a forkhead DNA binding domain, a leucine zipper, a zinc finger, and a polyglutamine tract. Based on secondary structure analysis, the DNA-binding domains of FOXP1, FOXK1a, and FOXK1bmay constitute a subfamily in the winged helix/forkhead family and consist of four a-helices and three b-strands arranged in the order H1-T1-S1-H2-H3-S2-W1-S3-H4. In this study we expressed the DNA-binding domain of FOXP1 (residues 463-565) in E. coli and purified it to homogeneity. The molecular weight and the DNA-binding activity of the DNA-binding domain of FOXP1 have been examined by mass analysis and gel mobility assay. The structural and dynamic studies of the DNA-binding domain of FOXP1 are ongoing.




The effect of phosphatidylglycerol on the organization of LHC II trimer

Jing Leng, Hui Chen, Liangbi Li *

Photosynthesis Research Center, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China


The light-harvesting chlorophyll a/b-protein complex of photosystem II(LHC II) is the most abundant pigment protein complex in green plants. The complex mainly serves as antenna to capture and transfer light energy to photosynthetic reaction center, and participates in the regulation of excitation energy distribution between two photosystems, the formation of stacking grana in thylakoid membrane, light protection and adaptation to environment. Therefore it is very important to study the relationship between the structure and function of LHCII. It has been shown that LHC II mainly exists in trimeric form in the thylakoid membrane of higher plants. The organization of LHC II trimer needs membrane lipids, while there is less knowledge regarding contribution of the membrane lipids in structure and function to the formation of LHC II trimer at present.

In this study, LHC II was isolated from spinach chloroplasts by high-speed centrifugation after a non-ionic detergent treatment. Native and SDS denaturing polyacrylamide gel electrophoresis analysis indicated that the isolated complex was a heterotrimer and consisted of three polypeptides with molecular weights of 29 KDa, 28KDa and 26KDa, respectively. The determination of membrane lipid and fatty acid compositions in the isolated LHCII showed that the LHCII contained the same four membrane lipids (MGDG, DGDG, PG and SQDG) as photosystem II. But the content of PG (phosphatidylglycerol) with unusual 16:1-trans-hexadecenoic acid as the predominant fatty acid chains was two times of that in photosystem II. The treatment of the isolated LHCII with phospholipase A2(PLA2) induced the dissociation of the complex to monomer. Re-formation of trimer from the monomer happened after adding PG into the dissociated LHCII. This phenomenon showed that PG was directly involved in the trimer organization and stabilazation. In addition, the absorption band at 475nm, 655nm and the fluorescence excitation peak at 480nm decreased obviously in the monomer, indicating that the energy transfer from Chl b to Chl a in the LHCII was effected after deletion of PG. From above results, it was suggested that PG not only played an important role in LHCII trimer formation and stabilization but also effected the pigments binding and the energy transfer within the LHCII.




Characterization of a Nonselective Cation Channel Prepared from

Tomato Roots

Dae-Seop Shin* and Young-Kee Kim

Department of Agricultural Chemistry, Chungbuk National University,

Gaeshin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Korea


In order to characterize the ion channels of tomato root cells, microsomes were prepared from the root tissue and were incorporated into the artificial lipid bilayer. Five types of ion channel were identified and their electrical conductances were 800, 500, 450, 265, and 155 pS. A nonselective cation channel(TNSC) was frequently found and its conductance was 450 pS. The TNSC had three subconductance states and their conductances were 450, 257 and 105 pS. All conductance states showed linear current vs. voltage relationships. At positive holding potentials, transient channel openings of high frequency gating between close and open states were observed. However, at negative potentials, the channel stayed open for long time and open probability(Po) was 0.83 at -40 mV. Ion selectivity of the channel was measured at the asymmetric ion condition. When additional 50 mM K+ and Na+ were added to the cis side of bilayer, the reversal potentials were shifted to negative potentials and obtained the same potentials of -10 mV. These results imply that TNSC is a nonselective cation channel. The effects of various heavy metal ions were investigated at the concentration of 100 μM. The channel activity was severely inhibited by La3+, Ba2+, and Zn2+, and Po was decreased to less than 0.2 at negative holding potentials. However, Al3+ and Cd2+ decreased the activity by 20%. Interestingly, each metal ion showed different kinetics of channel inhibition. While La3+ inhibited the activities of all subconductance states at 500 μM, Zn2+ at 1 mM inhibited all subconductance states except 105 pS. Cd2+ changed the gating of TNSC form long-opening state to transient openings even at negative holding potentials. These data represent that those metal ions have different binding sites on the channel protein and they can be useful modulators to investigate structural characteristics as well as the functional roles of TNSC on the root physiology.




Effects of Organic Compounds on the Tolaasin-induced Hemolysis

Young-Kee Kim* and Kwang-Hyun Cho

Department of Agricultural Chemistry, Chungbuk National University,

Gaeshin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Korea


Tolaasin, a Pseudomonas peptide toxin, causes brown blotch disease on the cultivated mushrooms by forming membrane pores and thus collapsing cellular structure. To understand the molecular mechanism of pore formation, we characterized tolaasin-induced hemolysis by measuring the binding and pore formation of tolaasin molecules to erythrocytes. Tolaasin molecules are likely to multimerize before they bind to cellular membrane. Multimerization could be occurred by hydrophobic interactions of N-termini of the peptides because of the cationic nature of C-terminus. In order to suppress hydrophobic interaction of tolaasin, polarity of buffer solution was reduced by the additions of various organic solvents. Tolaasin-induced hemolysis was decreased by methanol and DMSO. However, other solvents, such as ethanol, buthanol, and glycerol, showed no effect on hemolysis. To investigate the inhibitory effects of organic solvents, two different experiments were done. First, the bindings of tolaasin molecules were measured in the presence of methanol and DMSO. Second, after erythrocytes were incubated with tolaasin, subsequent hemolyses were measured in the presence of methanol and DMSO. In the case of methanol, the preincubation of tolaasin with methanol did not changed time-dependent kinetics of hemolysis. However, when tolaasin was preincubated with DMSO, the hemolysis was severely delayed. These results show that DMSO inhibits the binding of tolaasin to plasma membrane, probably by disrupting the multimerization of tolaasin, while methanol inhibits the pore formation of membrane-bound tolaasin without any effect on the membrane binding of tolaasin.




Fluorescence Resonance Energy Transfer (FRET) Study on Interaction of

Heat Shock Protein hsp27 with p38 MAP kinase in Single Living Cell


Chunlei Zheng1, Ziyang Lin2, Yajun Yang1, Hanben Niu2 and Xun Shen1*

1Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China and

2 Institute of Optoelectronics, Shenzhen University, Shenzhen 518060, China


The stress response small heat shock protein 27 (hsp27) helps the cell in repair processes after environmental stress such as heat, UV-irradiation and oxidative stress. MAP kinase p38 is often activated by stress and various cytokines. It has been suggested that the activated p38 can phosphorylate MAPK-activated protein kinase 2 (MK2), then, the later phosphorylates hsp27. However, it has not been clear if there is any direct interaction between hsp27 and p38. In this study, we demonstrate a direct interaction between these two proteins by using fluorescence resonance energy transfer (FRET) technology in single living cell.

Two vectors expressing CFP (cyan fluorescence protein)-hsp27 fusion protein and YFP (yellow fluorescence protein)-p38 fusion protein were co-transfected into L929 cells. All FRET microscopic observations were performed on a Leica confocal laser scanning microscope system at 37 °C 12 h after transfection. Time-correlated single photon counting fluorescence lifetime imaging (FLIM) was also performed to obtain the donor CFP fluorescence lifetime images in the cells in the presence and absence of YFP-p38 respectively.

It was found that the fluorescence intensity of donor CFP significantly decreased in the presence of acceptor YFP, while the fluorescence of the YFP-p38 fusion protein induced by energy transfer from CFP was markedly enhanced by the presence of the CFP-hsp27 fusion protein. Photobleaching of the acceptor YFP led to about 50 % increase of the fluorescence of the CFP-hsp27 fusion protein, indicating at least 35 % excited energy of CFP in the fusion protein was transferred to YFP in the YFP-p38 chimera. The FLIM studies revealed that the lifetime of the CFP-hsp27 fusion protein was shortened from 2.27 ns in the absence of the YFP-p38 to 1.17 ns in the presence of the YFP-p38 chimera. The results suggest that the protein hsp27 and p38 are so close that the resonance energy transfer occurs between the two proteins. The effect of the p38 activation on the interaction of hsp27 with p38 was also investigated through stimulating the transfected cells with either UV-irradiation or hydrogen peroxide. It was interesting to notice that after UV-irradiation or H2O2-stimulation the fluorescence lifetime of the hsp27-fused CFP went up, indicating the weakening of the interaction between hsp27 and p38. The results imply that the FRET from CFP-hsp27 to YFP-p38 in living cells decreased when p38 was activated. Although it was reported that hsp27 exists in a signaling complex containing p38, MK2 and Akt, and the phosphorylation of hsp27 leads to its dissociation from the complex, it may need further investigation on the p38 activation-caused disruption of the interaction between hsp27 and p38.




A Radical Metabolism as A Partner of Energy Metabolism in Mitochondria

Xu Jian-xing

Institute of Biophysics, Chines Academy of Science, Beijing 100101 China


Mitochondria have been paid more and more attention since Wang et al found that the cytochrome c is involved in cell apoptosis [1]. Looking back to the history of mitochondria, it has been known for long time that the mitochondria not only produce ATP but also produce H2O2 [2]. In the past almost half century, the studies on mitochondria were mainly focused on the function of ATP synthesis. On the other hand the function of mitochondria producing H2O2 does not well promoted until Chance developed a method to detect H2O2 production in mitochondria [3]. Chance et al determined that about 2% of oxygen consumed in mitochondria was used in the H2O2 production in normal physiological condition [4]. This means that the H2O2 production is companioned with ATP synthesis in mitochondria.

How does the H2O2 production correlative to the respiratory chain?

What is the role of H2O2 production in mitochondria?

These years the production of H2O2 has been well studied. It has been proved that the precursor of H2O2 is the O2-. and O2-. is generated through a way of O2 single electron reduction by the leaked electrons from substrate-side of respiratory chain[5-8].

What is the way of H2O2 elimination in mitochondria? To answer this question is important because the O2-.and H2O2 will cause damage if they were not be eliminated in time.

I proposed a way of H2O2 elimination in 1995[9] by assuming that the cytochrome c of respiratory chain leaks electron to reduce H2O2, and pointed out that a radical metabolic path of O2-. ®H2O2 ®H2O is formed as a result of electron leakage of respiratory chain.

The key point for supporting this idea is to prove that the cytochrome c of respiratory chain can deliver electrons to the endogenesis H2O2. The following experimental evidence has been obtained in our lab recently.

1, Electrons transferred from succinate to cytochrome c in the presence of succinate-cytochrome c reductase can be further delivered to H2O2, no matter the H2O2 is extra added or generated by reductase itself. 2, The extraction of cytochrome c from HMP(a preparation for studying respiratory chain) not only cause the oxidase activity lost but also cause the generation of O2-.and H2O2 7-8 times higher increased. Reconstitution of cytochrome c to the cytochrome c depleted HMP causes the activity recovered and the O2-. H2O2 generation decayed exponentially. Similar behavior can also be observed in the cytochrome c depleted mitochondria. 3, Level of electron leak of respiratory chain is always higher when animal in pathological states, such as in aging and degenerative diseases.

A new version on the function of mitochondria can be obtained by assuming a radical metabolism as a partner of energy metabolism. The ATP production is needed but the H2O2 production contains a potential dangerous.




Molecular Mechanism of Hanatoxin Binding-Modified Gating in

Voltage-Gated K+-Channels

Kuo-Long Lou1*, Po-Tsang Huang2 and Yen-Chywan Liaw3

1Institute of Oral Biology, 2Inst. of Biochem. & Mol. Biology, Medical College, National Taiwan University,

Taipei 10042, Taiwan and 3Inst. of Molecular Biology, Academia Sinica, Taiwan

No.1 Chang-Teh Street, Taipei, 10042, Taiwan


While S4 is known as the voltage sensor in voltage-gated potassium channels, the carboxyl terminus of S3 (S3C) is of particular interests concerning the site for gating modifier toxins like hanatoxin. The thus derived helical secondary structural arrangement for S3C, as well as its surrounding environment, has since been intensively and vigorously debated. Our previous structural analysis based on molecular simulation has provided sufficient information to describe reasonable docking conformation and for further experimental designs. However, if one only relies on such information, more advanced structure-functional interpretation for the roles S3C may play in the modification of gating behavior upon toxin binding will remain unclear. In order to have better understanding on the molecular details regarding this issue, we have further performed the docking simulation with the S3C sequence from the hanatoxin-insensitive K+-channel, shaker, and analyzed the conformational changes resulting from such docking. Compared with other functional data from previous studies with respect to the proximity of S3-S4 linker region, we suggested a putatively significant movement of drk1 S3C, but not shaker S3C, in the direction presumably towards S4, which was comprehended as a possible factor interfering S4 translocation during drk1 gating in the presence of toxin. In combination with the discussions for structural roles of the length of S3-S4 linker, as well as stopped-flow results suggesting higher Koff for the aforementioned complex, molecular mechanism illustrating the hanatoxin binding-modified gating can be proposed.




Plasticity of Fitness and Diversification Process During an Experimental Molecular Evolution

*Akiko Kashiwagi1, Wataru Noumachi1, Masato Katsuno1, Mohammad T. Alam1, Itaru Urabe1, Tetsuya Yomo1,2,3,4

1Department of Biotechnology, Graduate School of Engineering, Osaka University,

 2Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 3Department of Pure and Applied Sciences, The University of Tokyo, , 4PRESTO, JST, JAPAN

1 2-1 Ymadaoka, Suita, Osaka, 565-0871 JAPAN, 22-1 Ymadaoka, Suita, Osaka, 565-0871 JAPAN, 33-8-1 Komaba, Meguro-ku,Tokyo 153-8902, Japan


A simplified experimental evolution encompassing the essence of natural one was designed in an attempt to understand the involved mechanism. In our system, molecular evolution was observed through three serial cycles of consecutive random mutagenesis of the glutamine synthetase gene and chemostat culture of the transformed Escherichia coli cells containing the mutated genes. Selection pressure was imposed solely on the glutamine synthetase gene when varieties of mutant genes compete in an unstructured environment of the chemostat. The molecular phylogeny and population dynamics were deduced from the nucleotide sequences of the genes isolated from each of the chemostat runs. An initial mutant population in each cycle, comprised of diversified closely-related genes, ended up with several varieties of mutants in a state of coexistence. Competition between two mutant genes in the final population of the first cycle ascertained that the observed coexisting state is not an incidental event and that cellular interaction via environmental nutrients is a possible mechanism of coexistence. In addition, the mutant gene once extinct in the previous passage was found to have the capacity to reinvade and constitute the gene pool of the later cycle of molecular evolution. These results, including the kinetic characteristics of the purified wild-type and mutant glutamine synthetases in the phylogenetic tree, revealed that the enzyme activity had diverged, rather than optimized, to a fittest value during the course of evolution. Here, we proposed that the plasticity of gene fitness in consequence of cellular interaction via the environment is an essential mechanism governing molecular evolution.




Evidence for Membrane Thinning Effect as the Mechanism for

Peptide- induced Pore Formation

Ming-Tao Lee1*, Fang-Yu Chen1 and Huey W. Huang2

1Department of Physics, National Central University, Taiwan 320

2Department of Physics & Astronomy, Rice University, Houston, Texas 77251

No. 300, Jung-da Rd., Jung-li City, Taoyuan, Taiwan 320, R.O.C.


Antimicrobial peptides have two binding states in a lipid bilayer, a surface state S and a pore-forming state I.  The transition from the S state to the I state has a sigmoidal peptide-concentration dependence indicating cooperativity in the peptide-membrane interactions.  In a previous paper (Chen et al., Biophys. J. 82:908-914, 2002) the transition of alamethicin was measured in three bilayer conditions.  The data were explained by a free energy that took into account the membrane thinning effect induced by the peptides.  Here the full implications of the free energy were tested by including another type of peptide, melittin, that forms toroidal pores, instead of barrel-stave pores as in the case of alamethicin.  The transitions from the S state to the I state were measured by oriented circular dichroism.  The membrane thinning effect was measured by x-ray diffraction.  All data were in good agreement with the theory, indicating that the membrane thinning effect is a plausible mechanism for the peptide-induced pore formations.  Interestingly, a sign change of a key parameter in the theory reflected a change from barrel-stave pores to toroidal pores.




SA Channel can be Activated by Cytoskeletal Tension in Endothelial Cells

Masahiro SOKABE1,2*, Kimihide HAYAKAWA2, Hitoshi TATSUMI1

1 Department of Physiology, Nagoya University Graduate School of Medicine,

2 ICORP Cell Mechanosensing, JST, 65 Tsurumai Nagoya 466-8550, Japan


Effects of mechanical stress in actin cytoskeleton on the stretch activated cation channel (SAC) present in the basal cell surface were examined in cultured human umbilical vein endothelial cells (HUVECs). We developed a method by which we can apply localized mechanical stimuli to the cell while recording intracellular Ca2+ transients at the basal cell surface. Fibronectin coated beads were attached to the apical cell surface, then focal adhesions, which were connected to the basal focal adhesions via actin fibers afterward, were formed underneath the beads. A localized mechanical stimulus could be applied to the basal focal adhesions through the actin fibers by displacing (0.1-1um) the attached bead with a piezo-driven glass capillary. Intracellular Ca2+ increases at the basal cell surface were measured with the fluorescent Ca2+ indicator fluo-3 excited with the evanescent light. Immediately after stimulation a rapid increase in Ca2+ concentration was observed around the basal focal adhesions. This Ca2+ transient was significantly reduced by external Ca2+ deprivation or by Gd3+, a potent blocker for SAC, suggesting that the Ca2+ transient was mediated by the activation of SAC. Cytochalasin D also inhibited the Ca2+ response. These results demonstrated that the mechanical force was transmitted to the basal focal adhesions via actin fibers to activate SAC. To directly prove this hypothesis we microinjected phalloidin coated beads into the cell and applied a traction force to the beads attached on stress fibers by the laser trapping technique while recording whole cell currents and intracellular Ca2+. A transient Ca2+ increase and inward current were observed just after the application of traction force to the bead, strongly suggesting that the mechanical stress in actin fibers directly activate SAC. The force required for a single SA channel activation was estimated as low as 1-2 pN. Ultra fast near field Ca2+ imaging resolved the Ca2+ influx spots across individual SA channels near basal focal adhesions. Simultaneous imaging of integrin molecules indicated that SA channels are located near integrin molecules as close as few hundreds nm. In conclusion actin fibers can act as a force transmitter to activate SA channels near focal adhesions.