Sunney I. Chan (陳長謙)

Hanna S. Yuan (袁小琀)

Chin Yu (余靖)

Shan-Ho Chou (周三和)

Carmay Lin (林小喬)

David Hsiao (蕭傳鐙)

Konan Peck (白果能)

Sheng-Fa Yu (俞聖法)

Chun-Hua Hsu (徐俊華)

Wei-Jyun Chien (錢偉鈞)

Ching-You Lu

Huan- shiun Hsu (許桓巽)

Wei-Chen Lin (林維政)

Pei-Wen Chao (趙珮雯)

Lou-sing Kan (甘魯生)

 

Sunney I. Chan (陳長謙)                 

The mechanisms of proton pumping in quinol oxidases

Sunney I. Chan, Institute of Chemistry, Academia Sinica, Nankang, Taipei, Taiwan 11529 and Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, CA 91125, USA

The principles of redox linkage in electron-driven proton pumps will be reviewed and applied to “proton pumping” in the quinol exidases.  It is shown how the coupling of electron-gating and proton-gating with redox-linkage is essentialto the operations of the proton pump, including the kinetic competence and the vectional requirement of the process.  The proton-pumping in two quinol exidases, mitochondrial cytochrome bc1 and E. coli cytochrome bo3, will be discussed withinthe framework of these concepts.

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Hanna S. Yuan (袁小琀)                

STRUCTURAL AND FUNCTIONAL ANALYSIS OF A HNH FAMILY OF ENDONUCLEASE- COLICIN E7 PROTEIN

Tzu-Ping Ko,1 Wen-Yen Ku,1 Meng-Jiun Sui,1 Yu-Wen Liu,1,2 Kin-Fu Chak2 and

Hanna S. Yuan.1

1Institute of Molecular Biology, Academia Sinica, and

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

Colicins are bacteria toxins secreted by Escherichia coli to kill other Escherichia coli.  The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein.  This biological event provides a good model system for the investigation of protein recognition.  We have been focusing on the structural study of a DNase-type colicin, colicin E7 (ColE7), which contains a DNase domain that it cleaves DNA in target cells leading to the cell death.  We previously determined the crystal structures of the immunity protein Im7 and the one-to-one complex between the DNase domain of ColE7 and Im7 at 1.8 Å and 2.3 Å resolution, respectively.  The complex structure explains why the two proteins interact with each other with such a high specificity and affinity. 

The structure of the DNase domain of ColE7 contains a HNH motif within which a zinc ion is identified and the zinc ion is bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. The characterization of the metal-dependent DNase activity of ColE7 shows that a divalent metal ion is required and the enzyme is most active with the presence of Mg2+ or Ni2+ as a cofactor.  The crystal structure of the phosphate-bound complex determined at 2.0 Å resolution further shows that a phosphate ion is bound to the zinc site.  This result suggests that the metal ion in ColE7 is involved in DNA-binding and it is likely that the endonuclease active site is located around the zinc-binding site.  However, this putative DNase active site is not directly blocked by the immunity protein.  We suggest that the immunity protein blocks the DNA-binding site or induces conformational change in the colicin so that a colicin cannot bind to DNA for hydrolysis.

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Chin Yu (余靖)                

NMR Studies on Newt (Notophthalmus viridescens) Acidic Fibroblast Growth Factor:  Structure, Dynamics, and Interactions.

Chin Yu , Chemistry Department, National Tsing Hua University, Taiwan

Newt acidic fibroblast growth factor (nFGF-1) plays an important role in several cellular processes including cell growth, cell differentiation, angiogenesis, wound healing and other chemotactic activities.  Recently, nFGF-1 was cloned and expressed in E. Coli. and the double labeld (15N and 13C) protein (nFGF-1) was prepared.  A series triple resonance experiments were carried out.  The NMR 3D structured of nFGF-1 were calculated by using distance-geometry calculation followed by the dynamical simulated annealing techniques.  The backbone dynamics studies on nFGF-1 were accomplished by using 15N relaxation and heteronuclear NOE measurements and data were analyzed by model-free approach.  Glycosoaminoglycans such as heparin and heparan sulfate are known to activate nFGF-1 by inducing the dimerization of the receptor tyrosine kinase and thereby triggering the signal transduction cascade.  Molecular level understanding of the binding of glycosoaminoglycans and its analogs to nFGF-1 is necessary for the design of drugs to inhibit nFGF-1 action.  A detailed NMR study using an uniformly 15N labeled sample has been carried out for newt nFGF-1 bound to sucrose octasulfate. From the chemical shift perturbation and intermolecular NOEs it was found that those residues belonging to the putative heparin binding domain spanning residues 126 to 142 are involved in the binding of sucrose octasulfate to newt nFGF-1.

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Shan-Ho Chou (周三和)               

Homo- and Heteronuclear NMR Studies of the Purine-rich and Pyrimidine-rich DNA Mini-Loop Hairpins

Shan-Ho Chou, Institute of Biochemistry

National Chung-Hsing University, Taichung, 40227, Taiwan

Nucleic acids are special biomolecules; they can serve as a catalyst, aptamer, or a carrier of heritable information. Due to the advancement of powerful techniques such as PCR (polymerase chain reaction) and SELEX (selected enrichment of ligand by exponential multiplication), nucleic acid sequences possessing any required function can now be screened out without much difficulty. Many novel functions for nucleic acids have therefore been discovered, including those involved in enzymatic reactions and monoclonal aptamer etc.

It is obvious that a linear single-stranded nucleic acid sequence can only execute its function when folded into a specific tertiary structure through hairpin formation. Besides, hairpins can also be existent when intra-strand pairing occurs between inverted repeat sequences in double-stranded DNA. It is therefore interesting to study such fundamental motifs.

While tetraloops of the type GNRA and UNCG are prevalent in the RNA structure, triloops are instead the major forms in the DNA structure. In the past, we have been studying purine-rich and pyrimidine-rich triloop hairpins and found they adopt dramatically different structures. The homo- and heteronuclear NMR studies of such compact stable motifs will be discussed, as well as their biological significance.

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Carmay Lim (林小喬)               

Metal Binding in Proteins:

The Effect of the Dielectric Medium, Ligand Type and Geometry

Todor Dudev and Carmay Lim*

Institute of Biomedical Sciences, Academia Sinica,

Taipei 11529, Taiwan, Republic of China.

This work attempts to addresses three aspects of metallo-protein chemistry:  First, why metals ions tend to bind to proteins directly (in an “inner-sphere” mode) at centers of high hydrophobic contrast? Second, what is the most thermodynamically preferable set of inner-sphere ligands for a given metal ion (e.g. magnesium) in proteins? Third, what is the most thermodynamically preferable coordination geometry of a given metal ion (e.g. zinc) in proteins? To address these questions, a combination of ab initio and continuum dielectric calculations as well as Protein Data Bank (PDB) surveys of metal-binding sites in proteins were carried out. The results show that a low dielectric medium favors the inner-sphere binding of protein ligands to the metal. Using magnesium as an example, the results delineate its most thermodynamically preferable inner-sphere set of inner-sphere ligands and suggest an explanation for the unique role of Mg2+ as a carrier of water molecules that mediate enzymatic hydrolysis reactions. Finally, using zinc as an example, the results show that zinc prefers to be tetracoordinated in a low-dielectric protein environment, hence the tetrahedral zinc binding sites are not necessarily energized states, as proposed in earlier works.

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David Hsiao (蕭傳鐙)              

Structure and function of a moonlighting protein: phosphoglucose isomerase/ autocrine motility factor/neuroleukin/differentation and maturation mediator

Chwan-Deng Hsiao1 , Yuh-Ju Sun1, Chia-Cheng Chou1, Wei-Shone Chen2, Rong-Tsun Wu3, and Menghsiao Meng4 1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan. 2Veterans General Hospital, Taipei, Taiwan. 3Graduate Institute of Biopharmaceutial Science, National Yang-Ming University, Taipei, Taiwan. 4Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan.

 

        Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways.  PGI has been found to serve functions equivalent to those of autocrine motility factor (AMF), a tumor-secreted cytokine which stimulates cell migration in vitro and metastasis in vivo, and to those of neuroleukin (NLK), a neurotrophic factor for spinal and sensory neurons.  Here we present the first complete crystal structure of phosphoglucose isomerase at 2.3 _ resolution from Bacillus stearothermophilus.  We also demonstrate that PGI performs cell motility-stimulating activity on mouse colon cancer cells similar to that of endogenous AMF, and that PGI enhances neurite outgrowth in terms of length and density on neuronal progenitor cells similar to that observed for NLK.   The results confirm that PGI is NLK and AMF.  The structure of PGI is a open twist a/b structure motif consisting of two globular domains and two protruding parts.  Based on this substrate-free structure, together with the biological, biochemical and modeling results, we postulate the possible substrate binding-site located within the domains’ interface for PGI/AMF based on previous observations that both proteins share the same inhibitor site.  The structure further provides evidence to suggest that the top part of the large domain and the protruding loop of PGI/NLK play roles in inducing neurotrophic activity.

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Konan Peck (白果能)             

DNA chips and Their Applications in Genomic Biotechnology

Konan Peck, Yuh-Pyng Sher, Ji-Yen Cheng and Yun-Shien Lee

Institute of Biomedical Sciences, Academia Sinica

Taipei, Taiwan 115, ROC

DNA microarrays or DNA chips are the most recent technology breakthroughs in genomic studies. The methods allow genome-wide analysis of thousands of genes at a time. Two high capacity systems based on oligonucleotide and cDNA microarray approaches and colorimetric detection method (Microarray/CD) are developed. The oligonucleotide microarray system was designed for typing single nucleotide polymorphism and genetic variations. The cDNA microarray system was designed to simultaneously monitor the expression of thousands of genes in human cells treated with and without external stimuli.

For the oligonucleotide microarray fabrication, a high-capacity multiplex oligonucleotide synthesizer was constructed to simultaneously synthesize 384 discrete oligonucleotides in about 10 hours. The synthesis was based on phosphoramidite chemistry with step yield routinely over 98.5%. The oligonucleotide probes were spotted in microarray format on Nylon filter membranes by an arraying machine developed in-house. The chemistry for immobilizing the oligonucleotide probes on Nylon filter membranes and the protocol for genotyping single nucleotide polymorphisms in a gene have been developed.

For the cDNA microarray system, the target genes were selected based on an in-house constructed UniClone database containing the longest EST clone for every gene cluster built by the NCBI Unigene clustering. Based on the UniClone compilation, an indexed cDNA clone set containing thousands of non-redundant gene clones has been constructed for genome-wide gene expression studies. To array thousands of non-redundant cDNA fragments on Nylon filter membranes, a high capacity arraying machine capable of holding 31,000 of PCR products at a time and depositing 31,000 spots on a piece of 1.8 cm by 2.7 cm Nylon membrane have been developed. Several on-going applications of the cDNA microarray system implemented in our lab will be described.

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Sheng-Fa Yu (俞聖法)               

DIOXYGEN ACTIVATION AND ALKANE HYDROXYLATION BY THE MEMBRANE-BOUND METHANE MONOOXYGENASE IN METHANOTROPHIC BACTERIA

Sheng-Fa Yu1, Sean J. Elliott2, and Sunney I. Chan1,2

Institutee of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan1

A.A. Noyse Laboratory of Chemical Physics, California Institute of Technology, Pasadema, CA 91125, U.S.A.2

The particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the conversion of methane to methanol in methanotrophic bacteria.  The pMMO isolated from Methyloccocus capsulatus (Bath) consists of a three-subunits hydroxylase (45, 27 and 23 kDa) and an NADH oxidoreductase (38 kDa).  The hydroxylase is a copper protein, with 15 copper ions arranged in five trinuclear copper clusters.  Two of the copper clusters are associated with the 27 kDa subunit and are involved in the hydroxylation chemistry of the enzyme.  The remaining three copper clusters are associated with the 45 kDa subunit, provide a buffer of reducing equivalents in the protein, and participate in electron transfer, mediating the electron input from the NADH reductase to the catalytic clusters.  On the basis of chemical and proteolytic treatments, the bulk of the copper ions are localized in the exposed water-soluble domains of the proteins.  The remaining copper ions (ca. 6) are sequestered in the membrane-associated domain.  The functional form of the enzyme is the fully or partially reduced copper hydroxylase.  The pMMO exhibits unusual substrate specificity: only small normal alkanes are hydroxylated and similar alkenes epoxidated.  In addition, the chemistry is highly regiospecific as well as stereoselective.  It is apparent that the substrate binds to a short and narrow hydrophobic pocket, and the hydroxylation chemistry involves concerted binding of the C-H bond to an activated copper cluster and insertion of the C-H bond to an activate copper cluster and insertion of the “active” oxygen species.  Further evidence in support of the stereoselectivity of the hydroxylation chemistry is presently sought by experiments involving a series of designed deuteriated chiral alkanes as substrates.  A mechanism for the dioxygen activation mediated by the catalytic clusters will be proposed.

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Chun-Hua Hsu (徐俊華)               

THE SOLUTION STRUCTURES AND STRUCTURE- FUNCTION STUDIES OF RIBONUCLEASES FROM Rana catesbeiana (BULLFROG) WHICH POSSESS DIFFERENT RIBONUCLEOLYTIC ACTIVITY AND CYTOTOXICITY

Chun-Hua Hsu1,Ning-Yuan Su2,Cindy C-J Kao2,Shih-Hsiung Wu1,3 ,

,,,,     You-Di Liao2,Chinpan Chen2

[1] Institute of Biochemical Sciences, National Taiwan University, [2] Institute of Biomedical Sciences,  [3] Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, R. O. C

The NMR solution structure of RC-RNase, a pyrimidine-guanine sequence specific ribonuclease isolated from Rana catesbeiana (bullfrog) oocytes, was determined two years ago [ref 1,2].  In addition to possessing ribonucleolytic activity, RC-RNase is cytotoxic and can inhibit the growth of tumor cells, which is similar to onconase protein. The unique cytotoxic and antitumor activities of onconase and RC-RNase are now being exploited for their medical applications [ref 3].  Onconase is currently undergoing phase III clinical test as an antitumor drug and the potential of RC-RNase as anticancer drug is also under evaluation. Recently, Dr. You-Di Liao has isolated five more new ribonucleases (~ 105 residues for each protein) from oocyte and liver of Rana catesbeiana and checked their biological functions.  These new ribonucleases together with RC-RNase, RNase A as well as Onconase possessed different base specificity, ribonucleolytic activity and cytotoxicity.  They all belong to the RNase A superfamily.  In order to elucidate the structure-function relationships on these ribonucleases from Rana catesbeiana, several biophysical methods will be applied.  First of all, the Molecular Modeling have been carried out. Strikingly, we observe the charge distributions are related to activities of these RNases. Furthermore, CD experiments are used to check the protein stability, secondary structure, structural change at different pHs, and binding studies of ribonuclease with substrate analogues. NMR studies including structure determination, protein folding, and dynamics on these new ribonucleases using native and 15N and/or 13C-labeled proteins are being carried out.  The preliminary structural studies based on Modeling, CD, and NMR experiments will be presented in this poster.

References:

1.Chen, C.P., Hom, K., Huang, R.F., Chou, P.J., Liao,Y.D. and Huang, T.H. The secondary structure of a pyrimidine-guaninesequence-specific ribonuclease possessing cytotoxic activity from the oocytesof Rana catesbeiana.

J. Biomol. NMR 8: 331-344, 1996.

2.Chen, C.-F., Chen, C.P., Chen, Y.C., Hom, K., Huang, R.F. and Huang, T.H.. "The Solution Structure of a cytotoxic Ribonuclease from the Oocytes of Rana catesbeina (Bullfrog)". J. Mol. Biol. 283, 231-244 1998.

3. Huang, H.C., Wang, S.C., Leu, Y.J., Lu, S.C. and Liao, Y.D. The Rana catesbeiana rcr gene encodinga cytotoxic ribonuclease : Tissue distribution, cloning, purification,cytotoxicity, and active residues for RNase activity. J. Biol. Chem. 273:6395-6401, 1998.

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Wei-Jyun Chien (錢偉鈞)               

THE APPLICATION OF PFG-NMR SPECTROSCOPY ON THE DETERMINATION OF BINDING CONSTANT OF A CYCLIC PEPTIDE TO ANIONIC MICELLE

Wei-Jyun Chien and Gu, Li-Shiou

Department of Applied Chemistry, Chaoyang University of Technology

168 Gefeng E. Rd., Wufeng, Taichung, Taiwan, R.O.C.

Pulsed-field gradient (PFG) NMR spectroscopy has been used to measure the translational diffusion coefficient (Ds) of a cyclic peptide, SRIF14, under anionic micellar environment. Ds value was directly determined by following the decay of the resonance intensity of selected peptide proton. Using a two-state model, the association constant of SRIF14 to the micelle can be estimated. The obtained association constant of SRIF14-micelle binding constant is comparable to those measured from similar systems using other methods.

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Ching-You Lu              

Impairment of Free Radical Scavenging Enzymes and Enhanced Oxidative Damage in Skin and Muscle Fibroblasts from Patients with the CPEO Syndrome

Ching-You Lu, Edward-K. Wang, Hsin-Chen Lee, and Yau-Huei Wei

Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University and Section of Peripheral Neurology, Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan

Chronic progressive external ophthalmoplegia (CPEO) syndrome is characterized by ptosis and muscle weakness.  Mitochondrial DNA (mtDNA) analysis revealed that CPEO syndrome is associated with large-scale deletions or, less frequently, point mutations of mtDNA in affected tissues of the patients.  The molecular mechanism leading to mtDNA mutations is still obscure and the severity and onset age of the CPEO syndrome are poorly correlated with the proportion of mutant mtDNA in the patient's tissues.  It is thus of great interest to know whether there are some other defects that are involved in the pathogenesis of CPEO syndrome.  Recently, studies from this and other laboratories revealed that an imbalance between free radical scavenging enzymes occurs in a wide spectrum of human diseases and this may contribute directly or indirectly to the pathogenesis of the CPEO syndrome.  For studying the alterations of free radical scavenging systems in CPEO syndrome, skin and muscle fibroblasts from patients with CPEO syndrome were cultured.  We assayed the enzyme activities of Cu,Zn-SOD, Mn-SOD, catalase and glutathione peroxidase (GPx) of the fibroblasts.  The results showed that the skin and muscle fibroblasts from nine CPEO patients all had higher enzyme activities, protein and mRNA levels of Mn-SOD but those of catalase and GPx were not increased or even decreased.  These results indicate an imbalance between the H2O2 generation and removal systems in the fibroblasts from the nine patients with CPEO syndrome.  Moreover, we monitored the production of superoxide anion and H2O2 by flow cytometry and found that both levels in muscle fibroblasts from CPEO patients were higher than those of healthy controls.  The imbalance was much more profound in muscle fibroblasts, which is consistent with the clinical observation that muscle is the main affected tissue of patients with CPEO syndrome.  Take together, we suggest that in addition to the impairment of respiratory function the imbalance among free radical scavenging enzymes may play an important role in the pathogenesis and age-dependent progression of the CPEO syndrome.

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Huan- shiun Hsu (許桓巽)                  

A STUDY OF TRIPLE STRANDED DNA HELIX FORMATION WITH PSEUDOISOCYTIDINE BY USING THYMIDYLATE SYNTHASE MOTIF AS A TARGET

Huan- shiun Hsu1,3, R. Dayal Yadav1, Pei-Wen Chao1, Shwu-Bin Lin2,Tsung-Mei Chin3, Wei-Chen Lin1, and Lou-sing Kan1

1Institute of Chemistry,Academia Sinica,

2Graduate Institute of Medical Technology, National Taiwan University

3Graduate Institute of Chemistry,Chinese Culture Unveristy, Taipei, Taiwan

The thymidylate synthase (TS) catalyzed reaction is the sole de novo source of methylate of 2’-dexoyurine-5’-monophosphate (dUMP) to thymidine-5’-monophosphate (dTMP).  This reaction is required for DNA biosynthesis.  Because of its role in cell growth, TS has been a target of antineoplastic chemotherapy.  In this paper, we are exploring the inhibition of TS function by antigene triple stranded helix (triplex) formation.  We synthesized an oligodeoxyribonucleotides with a sequence in TS mRNA (5’-TTGGGGGGGGACCATGG-3’.(-13~7)) as a target for triplex formation.  This motif contains the starting codon of TS.  We designed two oligodeoxyribonucleotides, 5’-TCCCCCCCCAA-3’ and 5’-CCCCCCCCT-3’ for Watson-Crick and Hoogsteen base pairs to the target sequence, respectively.  In order to facilitate the triplex formation, we substitute the C in Hoogsteen strand by either 2’-O-methypesudocytidine (O) or 5methycytidine (M).  For this reason, two more oligomers, 5’-OOOOOOOOT-3’ and 5’MOMOMOMOT-3’, were synthesized

  The preliminary result showed that melting temperatures (monitored by UV thermodynamic study) of triplex formation are 15 /50 ; 24 /49 ; and 46.5 /46.5 for 5’-OOOOOOOOT-3’ as the Hoogsteen strand in solution of 0.15M NaC l, 0.02M MgCl2 with buffer at pH 7, 6, and 4, respectively.  On the other hand, the melting temperatures are 17/50; 27/48; and 45/45 for 5’-MOMOMOMOT-3’ for the same conditions.  The triplex Tm(s) are at pH 7,6 and 4 respectively for 5’-OOOOOOOOT-3’.  The two-stage melting warranties the triplex formation.  The kinetic studies of aforementioned triplexes formation are currently studied by surface Plasmon resonance.  (Supported by National Science Council, The Executive Yuan)

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Wei-Chen Lin (林維政)                  

A PRACTICAL LOOK OF SELCON - A PROGRAM FOR PROTEIN SECONDARY STRUCTURE ANALYSIS FROM CIRCULAR DICHROISM

Wei-Chen Lin and Lou-sing Kan

Institute of Chemistry, Academia Sinica, Taipei, Taiwan 11529

A program call SELCON that has been written by Sreerama and Woody in FORTRAN (Anal. Biochem., 209, 32-44 (1993)) is made for the initial guess for the secondary structure of proteins by circular dichroism spectra.  This paper presents a detail analysis of SELCON and step by step for how to using it to predict the unknown protein structure.  The modified program is available on inquiry and can be run on a IBM or IBM compitable personal computer. (Supported by National Science Council, The Executive Yuan)

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Pei-Wen Chao (趙珮雯)                 

STUDIES ON THE TAUTOMER OF PSEUDOISOCYTIDINE

Wei-Chen Lin, R. Dayal Yadav, Pei-Wen Chao, and Lou-sing Kan

Institute of Chemistry, Academic Sinica, Taipei, Taiwan 11529

The Pseudoisocytinide, owing to its potent anti-tumor activity, has attracted both chemists and biologists from the days of its inception. From structurally, it can be represented in two tautomeric form, one featuring an imino group (K1; meaning H is attached to N1) and another one fearing an imido group (K3, H attached to N3). From our theoretic calculation (Nucleosides & Nucleoides, 1999,18,1091-1093.) show that K1 isomer is less stable than K3 isomer by ca. ~2 kcal/mol in solution phase. Thus the proton exchange between the two tautomers is so fast that can not easy to get K1 and K3 separately.

Recently, we established a novel method for synthesis K1 and K3 tautomeric separately.  In order to reveal the imido proton exchange mechanism between K1 and K3, the proton and carbon-13 NMR titration experiment for each other have been carried out from pH 1 to 12 in aqueous solution. In contrast, K1 and K3 are not separable which is due to the fast exchange of the imido proton between N1 and N3 sites in aqueous solution, which observations are in agreement with our theoretical calculations. Furthermore, We found that the line broadening of C2 from carbon-13 spectra from pH 3 to 8 range under pH titration experiment. The phenomenon is involved in the imido proton exchange between N1 and N3 sites by through the C2 site. The exchange mechanism of the proton rests at N1 and N3 of pseudoisocytidine would be pointed out in this poster.

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Lou-sing Kan (甘魯生)               

PROTON NMR STRUCTURAL STUDIES OF 5’-d-(TC)3(CT) 3(AG) 3-3’ - A “Paper Clip” TRIPLE STRANDED HELIX WITHOUT LOOPS

Lou-sing Kan1, Laura Pasternack2, Shwu-Bin Lin3, T.-M. Chin4, Wei-Chen Lin1, and Dee-Hua Huang2

1Institute of Chemistry, Academia Sinica, Taipei Taiwan 11529

2Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037

3The Graduate Institute of Medical Technology, National Taiwan University, Taipei, Taiwan 10002

4Institute of Applied Chemistry, Chinese Culture University, Taipei, Taiwan 11114

A DNA octadecamer, 5’-T1C2T3C4T5C6C7T8C9T10C11T12A13G14A15G16A17G18, was found to form a “paper clip” type triple stranded helix (triplex) with the Hoogsteen strand binding in the major groove of the Watson-Crick duplex.  Proton NMR studies were conducted in aqueous solution at pH 4.5.  We find that the four internal base triads (C2+G14C11, T3A15T10, C4+G16C9, and T5A17T8) form typical pyrimidine-purine-pyrimidine hydrogen bonding configuration except the C2+ base is slight out of the plane.  At one end of the triplex, the bases C6+, C7, and G18 form a distorted but stable triad.  In contrast, at the other end of the triplex, T12 and A13 form a hairpin turn while T1 is stabilized by its association with T12-A13.  This is the first report of a stable triplex formed without loops. (Supported by National Science Council, The Executive Yuan)

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