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科技部化學推動中心補助學術研討會成果報告表
會議名稱：磁共振技術結合物理化學技術於生物分子應用研討會
舉辦日期：民國 106 年 5 月 19 日 至 民國 106 年 5 月 19 日
主辦機構： 清華大學生命科學院 主持人： 蘇士哲
舉辦地點： 高雄市義守大學行政大樓十樓 聯絡電話： 03 5742025
出席人數：工業界 30 人、學術界(含學生) 200 人 共 230 人
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會議重要成果： (如篇幅不足,另以 A4 白紙填寫)
1. 會議經過及議程
為促進台灣核磁共振研究之交流與提昇相關學術研究水準，每年在化學推動中心協助下，舉
辦核磁共振相關技術研討會，邀集國內外優秀研究學者一同與會並發表演講，提升該領域的
研究能量。本次研討會定名為“磁共振技術結合物理化學技術於生物分子應用研討會”，會
議時間為 2017 年五月十九日，於高雄義守大學舉辦，研討會主辦人為清華大學生命科學院兩位老師，一位為蘇士哲副教授（本計畫聯絡人，前任台灣磁共振學會秘書長，現任化學推
動小組核磁共振小組召集人）及呂平江教授（前任台灣磁共振學會理事長，現任台灣生物物
理學會理事長），此次會議安排是與台灣生物物理年會（五月十七至二十日）同一地點平行舉
行，但議程適度錯開。因為台灣生物物理年會是台灣最大的生物物理相關會議，與會人數達 350人，藉由同地舉辦，我們吸引台灣生物物理學界的學者加入討論核磁共振應用，與現有核磁共振學者互動，發展跨領域結合的可能。本研討會邀請演講者包含台灣磁共振學者、生物
物理及物理化學相關學者，同時邀請到來自韓國相近領域的十位學者，一同與會。活動規劃
為一整天，合計包含十六個演講，含蓋四個議題：先進核磁共振技術，分子結構研究，膜蛋
白研究及跨領域技術結合等，會議也邀請陳金榜研究員（中央研究院，現任台灣磁共振學會
理事長）及蔡明道院士擔任主持人，韓國兩位代表 Weontae Lee 教授（前任南韓生物物理學會理事長）及 Yangmee Kim 教授則是南韓核磁共振界相當活躍的學者。會議邀請的國內演講者，以具核磁共振使用經驗，能進行跨技術結合的學者為主，包含吳文桂教授，周三和教授，
黃聖言研究員等。因為與台灣生物物理年會結合，與會聽眾相當踴躍，合計產學研與會人數
超過 200人。詳細議程如後，同時附件為當日發送的議程手冊。
2. 國外講員其他演講行程
我們邀請到來自韓國的十位學者，於會議結束後進行一日的參訪，包含義守大學，佛陀紀念
館，中山大學等，隔日則陸續由小港機場離台，此次因無對國外講者進行機票及生活費補助，
所以無安排其他演講行程。
3. 重要收獲及心得
在過去十年間，先進核磁共振技術已成為研究生物分子不可或缺的主力，研究上除了可以提
供分子結構的訊息，同時也提供難得的分子動態及水溶液中的資訊。除了核磁共振技術本身
的優勢，未來如果想進一步研究更尖端的題目，必須結合到其他物理化學技術，來作進一步
的突破。今年我們擬與台灣生物物理學會合作，邀請該學會中具生物物理化學相關背景的學
者，一同舉辦一個交流性質的研討會，希望利用不同物化技術來與核磁共振相互結合，因為
與台灣生物物理年會結合，會議有將近 250 人與會，演講後的問答亦相當踴躍，足見會議交流相當成功。另外，此次研討會於南臺灣舉辦，會議也幫助於提升南部相關研究學者的參與，
並鼓勵南部學生對磁共振領域的了解與學習。
4. 建議
本次會議感謝化推中心的支持，讓小組會議有機會與生物物理年會聯合舉辦，讓生物物理學
會中具生物物理化學相關背景的學者有機會一同交流，使得核磁共振有機會做更廣泛的運
用，因為參與人數的增加，也讓實質效益大幅提升。將來其他小組也可以考慮做相對的聯合
會議。在舉辦會議經費上，限制可更放寬，如能簡化限制，也可以增加經費運用的彈性。例如演講必
須超過三十分鐘才能支領演講費，實際上，許多國際會議也都只進行二十分鐘的演講；大部份會議也都
會適度提供茶點，也可放寬申請；外國人無法支領演講費，在進行國際會議時，也不太方便。
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會議相關活動照片
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PROGRAM May 19, 2017 (Friday) 8:00-8:50 Registration 8:50-9:00 Opening remark Session A Chairman: Yuh-Ju Sun, National Tsing Hua University 9:00-9:30 TK1 Kyeong Kyu Kim, Sungkyunkwan University School of Medicine Noncanonical DNA: structure, function and modulation
9:30-10:00 TK2 Shan-Ho Chou, National Chung Hsing University
The Novel and Significant Roles Played by cyclic-di-GMP in the Microbial World
10:00-10:30 TK3 Yu-Chih Lo, National Cheng Kung University
The Recognition Mechanism of the Linear Ubiquitins by A20-Binding Inhibitors of NF-kB
10:30-10:50 Coffee Break Session B Chairman: Ming-Daw Tsai, Academia Sinica 10:50-11:20 TK4 Sangho Lee, Sungkyunkwan University
Molecular basis of ubiquitin recognition in DNA repair and linear polyubiquitin conformations by small-angle X-ray scattering
11:20-11:50 TK5 Hung-Wen Li, National Taiwan University
Single-molecule mechanistic studies on DNA homologous recombination
11:50-12:20 TK6 Tae-Young Yoon, Seoul National University Observation of single membrane proteins under mechanical tension
12:20-13:30 Lunch time (group photo)
Session C Chairman: Chwan-Deng Hsiao, Academia Sinica 13:30-14:00 TK7 Byung-Chang Suh, Daegu Gyeongbuk Institute of Science and
Technology (DGIST) Membrane phospholipid-dependency of CaV channel gating 14:00-14:30 TK8 Hyun-Ho Lim, Korea Brain Research Institute Stoichiometry of anion/proton coupling in CLC-type antiporters 14:30-15:00 TK9 Wen-guey Wu, National Tsing Hua University
Cobra Venom Actions Beyond Neurotoxicity 15:00-15:30 TK10 Ka-Young Chung, Sungkyunkwan University
Conformational mechanism of GPCR signaling analyzed by HDX-MS 15:30-15:50 Coffee Break Session D Chairman: Chinpan Chen, Academia Sinica
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15:50-16:20 TK11 Insuk So, Seoul National University School of Medicine
Dual action of the Gαq-PLCβ-PI(4,5)P2 pathway on TRPC1/4 and TRPC1/5 heteromultimer
16:20-16:50 TK12 Wei-hau Chang, Academia Sinica The essence of single-molecule imaging 16:50-17:20 TK13 Dennis W. Hwang, Academia Sinica
MRI Contrast Enhancement by Frequency Lock-in Technique 17:20-17:50 TK14 Yangmee Kim, Konkuk University Characterization of loop motions in protein by NMR spectroscopy 17:50-18:20 TK15 Yun-Ru Chen, Academia Sinica
Understanding TDP-43 Oligomers and Dipeptide Repeats in Frontotemporal Dementia and Amyotrophic Lateral Sclerosis
18:20-18:50 TK16 Chaok Seok, Seoul National University Prediction of protein complex structures by GALAXY 18:50-19:00 Close remark
磁共振技術結合物理化學技術於 生物分子應用研討會
19 May, 2017
I-‐Shou University, Kaohsiung No.1, Sec. 1, Syuecheng Rd., Dashu District, Kaohsiung City 84001, Taiwan, R.O.C.
Organizers: Biophysical Society of R.O.C. Department of Life Science, National Tsing Hua University I-‐Shou University Co-‐organizers: Life Science Research Promotion Center, Ministry of Science and Technology Chemistry Research Promotion Center, Ministry of Science and Technology Institute of Biological Chemistry, Academia Sinica Taiwan Protein Project, Academia Sinica Foundation of Health Sciences
http://biophys.sinica.edu.tw/Symposium/22BC/index.htm
TABLE of CONTENTS
Conference venue ................................................................... 1
Map ........................................................................................... 2
Program .................................................................................... 3
Abstracts .................................................................................. 5
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Date May 19th, 2017 Conference Venue 10F Conference room, Administration Building, I-‐Shou University No.1, Sec. 1, Syuecheng Rd., Dashu District, Kaohsiung City 84001, Taiwan, R.O.C. Photo Session 12:20-‐13:30, May 19th Scientific Program Committee • Carmay Lim, Institute of Biomedical Sciences, Academia Sinica • Nei-‐Li Chan, College of Medicine, National Taiwan University • Rita P.-‐Y. Chen, Institute of Biological Chemistry, Academia Sinica • Meng-‐Chiao Ho, Institute of Biological Chemistry, Academia Sinica • Hui-‐Chun Cheng, Institute of Bioinformatics and Structural Biology, National Tsing Hua
University, Taiwan • Shang-‐Te Hsu, Institute of Biological Chemistry, Academia Sinica • Chwan-‐Deng Hsiao, Institute of Molecular Biology, Academia Sinica • Yuh-‐Ju Sun, Institute of Bioinformatics and Structural Biology, National Tsing Hua
University, Taiwan Organizing Committee • Ping-‐Chiang Lyu, Institute of Bioinformatics and Structural Biology, National Tsing Hua
University, Taiwan • Jei-‐Fu Shaw, Department of Biological Science & Technology, I-‐Shou University, Taiwan • Shih-‐Che Sue, Institute of Bioinformatics and Structural Biology, National Tsing Hua
University, Taiwan • Chih-‐Hui Yang, Department of Biological Science & Technology, I-‐Shou University,
Taiwan • Hsien-‐Sheng Yin, Institute of Bioinformatics and Structural Biology, National Tsing Hua
University, Taiwan
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PROGRAM May 19, 2017 (Friday) 8:00-8:50 Registration 8:50-9:00 Opening remark Session A Chairman: Yuh-Ju Sun, National Tsing Hua University 9:00-9:30 TK1 Kyeong Kyu Kim, Sungkyunkwan University School of Medicine Noncanonical DNA: structure, function and modulation
9:30-10:00 TK2 Shan-Ho Chou, National Chung Hsing University
The Novel and Significant Roles Played by cyclic-di-GMP in the Microbial World
10:00-10:30 TK3 Yu-Chih Lo, National Cheng Kung University
The Recognition Mechanism of the Linear Ubiquitins by A20-Binding Inhibitors of NF-kB
10:30-10:50 Coffee Break Session B Chairman: Ming-Daw Tsai, Academia Sinica 10:50-11:20 TK4 Sangho Lee, Sungkyunkwan University
Molecular basis of ubiquitin recognition in DNA repair and linear polyubiquitin conformations by small-angle X-ray scattering
11:20-11:50 TK5 Hung-Wen Li, National Taiwan University
Single-molecule mechanistic studies on DNA homologous recombination
11:50-12:20 TK6 Tae-Young Yoon, Seoul National University Observation of single membrane proteins under mechanical tension
12:20-13:30 Lunch time (group photo)
Session C Chairman: Chwan-Deng Hsiao, Academia Sinica 13:30-14:00 TK7 Byung-Chang Suh, Daegu Gyeongbuk Institute of Science and
Technology (DGIST) Membrane phospholipid-dependency of CaV channel gating
14:00-14:30 TK8 Hyun-Ho Lim, Korea Brain Research Institute Stoichiometry of anion/proton coupling in CLC-type antiporters 14:30-15:00 TK9 Wen-guey Wu, National Tsing Hua University
Cobra Venom Actions Beyond Neurotoxicity
15:00-15:30 TK10 Ka-Young Chung, Sungkyunkwan University
Conformational mechanism of GPCR signaling analyzed by HDX-MS
15:30-15:50 Coffee Break
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Session D Chairman: Chinpan Chen, Academia Sinica 15:50-16:20 TK11 Insuk So, Seoul National University School of Medicine
Dual action of the Gαq-PLCβ-PI(4,5)P2 pathway on TRPC1/4 and TRPC1/5 heteromultimer
16:20-16:50 TK12 Wei-hau Chang, Academia Sinica The essence of single-molecule imaging 16:50-17:20 TK13 Dennis W. Hwang, Academia Sinica
MRI Contrast Enhancement by Frequency Lock-in Technique
17:20-17:50 TK14 Yangmee Kim, Konkuk University Characterization of loop motions in protein by NMR spectroscopy 17:50-18:20 TK15 Yun-Ru Chen, Academia Sinica
Understanding TDP-43 Oligomers and Dipeptide Repeats in Frontotemporal Dementia and Amyotrophic Lateral Sclerosis
18:20-18:50 TK16 Chaok Seok, Seoul National University Prediction of protein complex structures by GALAXY 18:50-19:00 Close remark
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Abstracts
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TK1 Noncanonical DNA: structure function and modulation Kyeong Kyu Kim Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Korea Most DNA in the genome is considered to be present as a B-form, but number of evidences support that DNA structures are highly polymorphic. Therefore, many sequence-specific noncanonical DNA (ncDNA) or non-B-DNA conformations transiently exist, often in response to changes in the cellular environment or when bound to proteins. A number of ncDNA structures, including triplexes, left-handed DNA, bent DNA, cruciforms, nodule DNA, flexible and writhed DNA, G-quartet (tetraplexes), slipped structures, and sticky DNA, have been discovered approximately one new conformation every 3 years for the past 35 years. ncDNAs are involved in various cellular events including chromatin remodeling, replication, transcription and recombination, and thus its presence or mutation is relevant to the various disease including tumors. In addition, it is verified that many genetic diseases are closely related to ncDNA. Therefore, it is considered that regulation of ncDNA formation is crucial to understand its cellular function and to develop a novel strategy for curing the related diseases. In this presentation, current progress of our studies on the structure, function and modulation of ncDNA will be introduced.
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TK2 The Novel and Significant Roles Played by cyclic-di-GMP in the Microbial World Shan-Ho Chou Institute of Biochemistry, National Chung Hsing University, Taichung, 40227, Taiwan, ROC The discovery of c-di-GMP second messenger was one of the most important breakthroughs in the microbial world in the past two decades. This molecule is present in most bacteria, regulating a plethora of important bacterial activities such as biofilm formation, biogenesis and function of flagella and pili, cell differentiation, and biosynthesis of natural product and secretion of pathogenic factors, through binding to an unprecedented array of effectors. There are usually tens or hundreds of enzymes that make or break c-di-GMP in every bacterial genome. However, only a few c-di-GMP receptors have been characterized to date. To get a better understanding of how c-di-GMP carries out its diverse functions, it is of crucial importance to decipher most or all possible c-di-GMP binding motifs. Several c-di-GMP receptors have been found but most of them usually exhibit narrow phylogenetic distribution1. Recently, MshE, an ATPase associated with the mannose-sensitive hemagglutinin type IV pilus formation in Vibrio cholerae, was shown to bind c-di-GMP well by a DRACALA methodology but no canonical binding motif was found in binding c-di-GMP. We have solved the crystal structure of the MshEN/c-di-GMP complex, which revealed an entirely new c-di-GMP binding mode2. It is fused with many other domains such as ATPase, glycosyltransferase, CheA, CheX, REC, cNMP-binding, HD-GYP, and guanylate cyclase, which have been found to play various important roles in bacterial physiology. MshEN is thus a new generation c-di-GMP binding protein that may serve as a good target for developing novel drugs against bacteria without causing drug resistance.
(1) Shan-Ho Chou* & Michael Y. Galperin* (2016) (Review) Diversity of c-di-GMP-binding proteins
and mechanisms, J. Bacteriology, 198 (1), 32-46. (2) Yu-Chuan Wang, Ko-Hsin Chin, Zhi-Le Tu, Jin He, Christopher J. Jones, David Zamorano
Sanchez, Fitnat H. Yildiz, Michael Galperin, & Shan-Ho Chou* (2016) Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain, Nature Communications, 7:12481.
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TK3 The Recognition Mechanism of the Linear Ubiquitins by A20-Binding Inhibitors of NF-kB Yu-Chih Lo1,2 , Shan-Meng Lin1, and Su-Chang Lin3 1Institute of Bioinformatics and Biosignal Transduction, National Cheng Kung University, Tainan, Taiwan 2Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan, Taiwan 3Genomics Research Center, Academia Sinica, Taipei, Taiwan Protein conjugation with ubiquitins is a critical biological process that mediates a variety of cellular events. The linear polyubiquitin is involved in non-degradative signaling pathways that mostly regulate the NF-kB activation, in which deubiquitinases play a significant role in deciphering ubiquitin codes for regulating signaling. More evidences in cell biology show that the linear polyubiquitin is also involved in tumor necrosis factor receptor (TNFR)-mediated cell death, as well as necroptosis. However, the molecular mechanism of how a polyubiquitin chain recognizing signaling molecules remains largely unknown. Emerging evidence in recent years shows that A20, a deubiquitinase, down-regulates the NF-kB activation signaling by interacting and degrading the polyubiquitin chains. The interaction is mediated by A20-binding inhibitors of NF-kB, ABINs, which contain both A20- and linear polyubiquitin interacting domains. Here we report our recent works on the ABIN2:polyubiquitin complex. Structural analyses together with the mutagenesis, pull-down, and isothermal titration calorimetry assays show that ABIN2 has a primary and a secondary linear ubiquitin-binding site. Surprisingly, a tri-ubiquitin molecule could simultaneously interact with two ABIN2 dimers, in which the ubiquitins form a helical trimer when bridging two hABIN2 dimers. Our studies suggest the formation of a higher-order complex between a linear polyubiquitin chain, ABIN2, and A20.
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TK4 Molecular basis of ubiquitin recognition in DNA repair and linear polyubiquitin conformations by small-angle X-ray scattering Sangho Lee Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Korea Small-angle X-ray scattering (SAXS) has been employed as a valuable tool to investigate the structure and conformational changes of biological macromolecules in solution. Ubiquitination attaches single or multiple ubiquitin moieties at specific residues such as lysine and methionine via isopeptide formation, generating various ubiquitin “codes”. Such ubiquitin codes are then utilized in regulating cellular processes ranging from proteasomal degradation to intracellular trafficking and DNA damage response. Rad18 is a key player in mediating DNA damage response signaling pathway where ubiquitination serves as platforms to recruit necessary factors in the pathway. Rad18 contains regions responsible for recognizing the ubiquitin “codes”. The Rad18:ubiquitin complex was investigated by SAXS to yield a structural model in solution. Subsequent biochemical and cellular assays validated the SAXS-derived structural model successfully. Rab5 is a master regulator in endocytic pathway. Rab5 can undergo ubiquitination in site-specific manners. SAXS-derived ubiquitinated Rab5 model suggested a molecular mechanism by which ubiquitination regulates the function of Rab5. Taken together, solution SAXS on proteins in ubiquitin-related pathways can yield valuable structural information from which our understanding of relevant biological processes is expanded.
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TK5 Single-molecule mechanistic studies on DNA homologous recombination Chih-Hao Lu1, Sheng-Yao Lin1, Peter Chi2 and Hung-Wen Li1 1Department of Chemistry, National Taiwan University, Taipei, Taiwan 2Institute of Biochemical Sciences, National Taiwan University and Academia Sinica, Taipei, Taiwan DNA homologous recombination is the major pathway to repair DNA double-strand breaks. Eukaryotic RAD51 and DMC1 recombinase are essential to this pathway in recognizing and pairing homology and carrying out strand exchange reactions. Recombinases first assemble onto single-stranded ssDNA to form nucleoprotein filaments. This filament assembly is the first and the committed step of homologous recombination and is subjected to tight regulation. During the assembly, nucleation step is kinetically slow, and several accessory proteins have been identified to stimulation nucleation. We have developed tethered particle motion and fluorescence resonance energy transfer methods to study the dynamics of recombinase nucleoprotein filament assembly at the single-molecule level. These single-molecule biochemical experiments allow us to determine and compare the size of recombinase nucleation cluster, nucleation rate, filament extension rate and filament stability in the presence of accessory proteins. Previous biochemical works showed that SWI5-SFR1 (S5S1), a heterodimeric accessory protein, interacts with RAD51, and stimulates RAD51-mediated homologous recombination. Our single-molecule experiments showed that mouse S5S1 interacts with mouse RAD51 to form complex, and the mRAD51-S5S1 complex efficiently stimulates the nucleation step. We also showed that mS5S1 stimulates mRAD51 nucleation by (i) reducing mRAD51 seed size, (ii) increasing ssDNA affinity and (iii) stabilizing mRAD51 nucleation cluster on ssDNA. Understanding different regulation strategies at the molecular basis allows engineering an efficient recombination progression.
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TK6 Observation of single membrane proteins under mechanical tension Tae-Young Yoon School of Biological Sciences, Seoul National University, Seoul 08826, Korea
Single-molecule methods, which handle and monitor molecules one at a time, have been a powerful toolkit for elucidating molecular mechanisms underlying many biological processes. I will talk about two recent efforts in my lab, where we endeavor to apply single-molecule methods to membrane proteins. First, by applying pN-scale force with magnetic tweezers, we revealed large hysteresis in a mechanical unizipping and rezipping cycle of the SNARE complex. With combined application of different single-molecule methods, we showed how this rigid SNARE complex is efficiently disassembled by NSF and SNAP. In particular, we observed that NSF exploits a spring-loaded mechanism to tightly couple its ATP hydrolysis with unfolding of the SNARE complex. Second, by employing single-molecule fluorescence imaging as the detection scheme, my lab has been developing single-molecule co-immunoprecipitation (co-IP) analysis. Harnessing single-molecule sensitivity and ms time resolution, we quantitatively determine the intensity of a cell signaling step encoded by corresponding protein-protein interactions (PPIs). This novel capability will shed light onto the molecular lesions that drive individual cancers at the PPI level, where signal transduction physically occurs.
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TK7 Membrane phospholipid-dependency of CaV channel gating Byung-Chang Suh Department of Brain and Cognitive Sciences, DGIST, Daegu 42988, Korea A growing body of data supports a view of the phospholipids of cell membrane as a key regulator of many ion channels and cellular excitability of neurons. The decrease in the proportion of polyphosphoinositides that occurs in response to the stimulation of G protein-coupled receptors (GPCRs) mostly reduces the open probability of ion channels and diminishes whole current size passing through the channel pores. Our research has been documented the functional mechanism of stimulus-induced phospholipid dynamics in ion channel regulation using a wide variety of experimental approaches. Most of the evidence suggests that normal ion channel activity requires membrane polyphosphoinositides, especially phosphatidylinositol 4,5-bisphosphate (PIP2). The molecular basis for the PIP2–dependent activation of ion channels and its physiological roles vary in diverse types of ion channels and cells. Here, I will introduce the molecular properties of phospholipid regulation in voltage-gated Ca2+ (CaV) channels.
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TK8 Molecular evolution of a membrane protein: CLC channel from/to CLC antiporter Hyun-Ho Lim Department of Structure & Functional Neural Network, Korea Brain Research Institute (KBRI), Daegu 41068, Korea Ion channels and ion transporters are essentially membrane-embedded enzymes, which catalyze the translocation of substrates (ions) in and out of cell membrane. However, these two proteins are operating with very different biophysical principles. In contrast to ion channels allow electrochemically downhill movement of ions upon stimulation, membrane transporters requires free energy input to transport ion in energetically uphill direction. Thus, it had been generally believed that these two are structurally and evolutionarily unrelated protein superfamily, until Chris Miller and his colleagues revealed that CLC family proteins turned out to be either Cl- channel or Cl-/H+ antiporter. The CLC superfamily can be found in virtually all organisms from bacteria to human. In mammal, genetic mutations in the CLC genes are linked to the various diseases such as myotonia, deafness, epilepsy, leukodystrophy, kidney malfunctions and lysosomal storage disease. CLC proteins also play key roles in the physiology of other organisms: nitrate uptake for nitrogen fixation in plants, gastric acid-resistance in enteric bacteria and fluoride (F-)-resistance mechanisms of unicellular microorganisms such as pathogenic bacteria and eukaryotic parasites. In E. coli, two CLC proteins, CLC-ec1 and CLC-ec2 are both functionally important for bacteria to survive extreme acid challenge: the double knockout cannot support survive at pH 2.5, but the presence of either CLC gene equally the double knockout mutant. However, in vitro purified and reconstituted CLC-ec2 shows much lower transport activity than CLC-ec1. How can CLC-ec2 have such a physiological contribution with such a functional difference? We have been collecting a line of evidences which suggests a likely answer: CLC-ec2 is activated below pH 3, where CLC-ec1 begins to shut down and CLC-ec2 activity is stimulated upon voltage changes to which CLC-ec1 does not respond. Moreover, CLC-ec2 swaps one proton with ~10 Cl- in the reconstituted proteoliposome, but the stoichiometry can be interestingly reduced to 1 to 4 (H+/Cl-) by transmembrane voltage stimulus and acidification. What can change the coupling ratio of transported ions in CLC-ec2? Systematic mutagenic approaches produce results that voltage-dependent H+ translocation could be the culprit of changing coupling ratio from. These results imply that the voltage-dependent H+ transport and the presence of Cl--slippage state might be the molecular determinants to build ‘ion channel’- and ‘transporter’-type CLC proteins. Currently, we are focusing on the structural determination of CLC-ec2 protein to obtain further structural and mechanistic insights.
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TK9 Cobra Venom Actions Beyond Neurotoxicity Wen-Guey Wu Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Taiwan, R.O.C Despite of the general belief that cobra venom actions mainly induce neurotoxicity of the bitten victims, recent clinical evaluation of cobra snakebites by Naja atra indicates a localized swelling and tissue necrosis as the most common symptom with less than 5% of the supposed block of neuromuscular junction. As the major toxin composition of Naja atra are CTXs (cardiotoxins/cytotoxins) capable of releasing ATP and NADH from the necrotic cells, we hypothesize that the metabolic products of the endogenous purinergic signaling pathway may play a significant role in the severe tissue necrosis. We then clone both phosphodiesterase (PDE) and nucleotidase cDNA from Taiwan cobra venom gland and purify them from crude venom to determine their 3D structure. The results indicate that they are respectively the structural homologues of human ecto-nucleotide pyrophosphotase phosphodiesterase (e-NPP1) and CD73. More interestingly, cobra PDE could also bind to the ectodomain of human insulin receptor (IR) to inhibit IR phosphorylation, followed by affecting on the m-TOR-related cellular activities. Our results suggest for the first time that cobra venom also consist of toxins capable of targeting glucose homeostasis to exhibit symptoms of muscle weakness and thirst, in addition to the previously well-known neurotoxic, inflammatory and thrombotic effect for the snakebite victims.
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TK10 Conformational mechanism of GPCR signaling analyzed by HDX-MS Ka Young Chung School of Pharmacy, Sungkyunkwan University, Suwon 16419, Korea Conformational change and protein–protein interactions are two major mechanisms of protein signal transduction. To understand the precise signaling mechanism, studies have investigated the structural mechanism of signaling proteins using X-ray crystallography, nuclear magnetic resonance (NMR), or electron paramagnetic resonance. In addition to these techniques, hydrogen/deuterium exchange mass spectrometry (HDX-MS) has recently been used in conformational analysis of signaling proteins. HDX-MS measures the rate at which peptide amide hydrogens exchange with deuterium in the solvent. Exposed or flexible regions have higher exchange rates and buried or ordered regions have lower exchange rates. Therefore, HDX-MS is a useful tool for studying protein–protein interfaces and conformational changes after protein activation or protein–protein interactions. Although HDX-MS does not give high-resolution structures, it analyzes protein conformations that are difficult to study with X-ray crystallography or NMR. Furthermore, conformational information from HDX-MS can help in the crystallization of X-ray crystallography by suggesting highly flexible regions. In this seminar, I will discuss the conformational mechanism of GPCR signaling as an example of signaling proteins analyzed by HDX-MS.
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TK11 Dual action of the Gαq-PLCβ-PI(4,5)P2 pathway on TRPC1/4 and TRPC1/5 heteromultimer Insuk So Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Korea The transient receptor potential canonical (TRPC) 1 channel is widely distributed in mammalian cells and is involved in many physiological functions. TRPC1 is primarily considered a regulatory subunit that forms heterotetrameric TRPC1/4 and TRPC1/5 channels to modify the pore properties of TRPC4 and TRPC5 and their activation by Gαq-coupled receptors. Here, we reveal that the self-limiting regulation of the heterotetramers by the Gαq-PLCβ pathway is dynamically mediated by PI(4,5)P2. We recorded channel activity and plasma membrane PI(4,5)P2 when manipulating Gαq activity and PI(4,5)P2 levels to conclude that, following G-protein coupled receptor (GPCR) activation, Gαq directly binds to TRPC1/4 and TRPC1/5 channels, resulting in channel gating. Simultaneously, Gαq-coupled PLCβ activation results in the breakdown of PI(4,5)P2. Dissociation of PI(4,5)P2 from the channels inhibits the activated currents. The subsequent increase in cytoplasmic Ca2+ due to Ca2+ release from the ER and activation of PKC resulted in a second phase of channel inhibition.
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TK12 The essence of single-molecule imaging Wei-hau Chang (章為皓 ) Institute of Chemistry, Academia Sinica, No. 128 Sec. 2 Academia Rd. Taipei, Taiwan With breakthrough of technologies on microscopy, camera and advanced light source, it has been demonstrated that biological molecules can be imaged at single molecule level. Nevertheless, the single-molecule data are much noisier than that observed by conventional bulk measurement. So, what is the benefit of seeing molecules as singles and what would be the challenges one person faces when he or she is committed to this field? As my lab has been engaging in various single-molecule imaging activities using microscopy, electron microscopy, or free-electron lasers, I will use our on-going projects to share the benefits and pitfalls of those experimental techniques and emphasize the key areas that demands further development. 隨著 顯微技術、 照相機的進步，還有新式光源的誕生，單一生物分子造影成為研究生物物理的重要手段。 然而，由單分子造影所得之資料與傳統集體測量方法所得之資料相比，往往訊躁比極低，以致分析困難。既然如此，為何還要從事向單分子這種性安價比極低的研究? 近五年以來，我們實驗室參加了跨領域計畫，必需要使用螢光顯微鏡、電子顯微鏡、和自由電子雷射從事單分子造影。我將會分享這些技術的優點和陷阱，以及其中亟需發展的部分。
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TK13 MRI Contrast Enhancement by Frequency Lock-in Technique Dennis W. Hwang Institute of Biomedical Sciences, Academia Sinica, Taiwan The goal of this study is to develop novel MR contrast by frequency lock-in technique. An electronic feedback device that generates a specific narrow frequency bandwidth RF field is presented. The effects of this RF field on magnetic resonance images are assessed both theoretically and experimentally. Spectroscopy and imaging experiments were performed. The first experiment utilized magnetizations with the same central frequency but different frequency distributions and was compared to MR images obtained with T2 contrast agents. Lock-in suppressed images showed an improvement in contrast relative to the conventional imaging method. The second experiment utilized magnetizations with small shifts in frequency and a broad frequency distribution. This is helpful for differentiating between small structural variations in biological tissues. The contrast achieved in in vivo tumor imaging using the lock-in suppressed technique provide higher spatial resolutions and discriminate the regimes of necrosis and activation consistent with pathologic results. In addition, Frequency tuning allowed the selected spectral peak to be suppressed. The contrast achieved in in vivo mouse brain imaging using the lock-in suppressed technique indicated a better spatial discrimination when compared with that achieved using conventional imaging methods, especially in the hippocampus region. Selective lock-in suppressed imaging is a new approach to provide frequency information in magnetic resonance imaging; rather than determining the evolution of image contrast over time, this approach allows small susceptibility variations to be distinguished by tuning the frequency of the narrow bandwidth lock-in RF field. A new and enhanced contrast can be achieved using this technique.
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TK14 Characterization of loop motions in protein by NMR spectroscopy Yeongjoon Lee, Min-Cheol Jeong and Yangmee Kim* Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea Flexibilities in protein conformations are essential for their functions, playing key roles in molecular recognition, rate-limiting conformational transitions, and catalysis. Phosphatases of regenerating liver (PRLs) constitute a novel class of small, prenylated phosphatases with oncogenic activity. PRL-3 is particularly important in cancer metastasis and represents a potential, therapeutic target. The flexibility of the WPD loop as well as P-loop of PRL-3 is closely related to its catalytic activity. Relaxation dispersion measurements provides a powerful approach for mapping the folding pathway and conformational exchange data provide the closing rate of the WPD loop between open and closed conformations. This intrinsic loop flexibility of phosphatase may be related to their catalytic rate and may play a role in the substrate recognition. Flexibility of acyl carrier protein (ACP) is essential for its ability to interact with functionally different enzyme partners in the rapid delivery of acyl chain from one partner to another. The overall structure of bacterial ACPs shows the common ACP folding pattern consisting of four α-helices connected by three loops. Compared to other ACPs, α2α3 loop in ACP from Enterococcus faecalis exhibits slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. Molecular details of loop movement in ACP provide insights into the ACP-mediated acyl chain elongation and might facilitate development of antibiotics against pathogenic drug-resistant strains.
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TK15 Understanding TDP-43 Oligomers and Dipeptide Repeats in Frontotemporal Dementia and Amyotrophic Lateral Sclerosis Yun-Ru Chen Genomics Research Center, Academia Sinica , Taiwan, R.O.C Inclusions comprising TDP-43 and dipeptide repeated (DPR) translated from hexanucleotide expansions in C9ORF72 gene were found in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The diseases affected different brain regions but have some overlapping symptoms. C9ORF72 hexanucleotide expansion can be translated to DPRs, including poly glycine-alanine (GA), glycine-arginine, glycine-proline, proline-arginine, and proline-alanine, which form inclusions in brain and spinal cord of C9ORF72-linked FTD/ALS patients. The structure and function of both TDP-43 and DPRs have not been elucidated. Hence, we employed biochemistry and cellular methods to examine TDP-43 oligomers and DPRs. We discovered full-length TDP-43 oligomers are present in brain of FTD-TDP patients. The full-length TDP-43 forms spherical oligomers that share common epitopes with amyloid oligomers. The TDP-43 oligomers are neurotoxic and capable to transform Alzheimer’s amyloid-β (Aβ) to Aβ oligomers. We further found TDP-43 interacts with Aβ through its N-terminal part. The effect is only shown when TDP-43 interacts with Aβ in the early stage. Meanwhile, we used synthetic poly (GA)15 DPR as a model system to examine its aggregation properties. We found that (GA)15 with 15 dipeptide repeats fibrillates rapidly to flat, ribbon-type fibrils. The fibrils bind to classic amyloid dyes and contain characteristic cross β-sheet structures. We also demonstrated that (GA)15 DPR is neurotoxic and capable of cell-to-cell transmission. Other DPRs are also under investigation. Overall, our results provide structural and toxicity properties of TDP-43 oligomers and GA DPR to facilitate future therapeutic development. (1) Yu-Sheng Fang, Kuen-Jer Tsai, Yu-Jen Chang, Patricia Kao, Rima Woods, Pan-Hsien Kuo,
Cheng-Chun Wu, Jhih-Ying Liao, Shih-Chieh Chou, Vinson Lin, Lee-Way Jin, Hanna S. Yuan, Irene H Cheng, Pang-Hsien Tu, and Yun-Ru Chen*. “Full-Length TDP-43 Forms Toxic Amyloid Oligomers that are Present in Frontotemporal Lobar Dementia-TDP Patients.” (*corresponding author) (2014) (Nature Communications, 5:4824 ).
(2) Patricia F. Kao, Yun-Ru Chen, Xiao-Bo Liu, Charles DeCarli, William W. Seeley, and Lee-Way Jin*. Detection of TDP-43 oligomers in frontotemporal lobar degeneration-TDP. (2015)(Annals of Neurology, 78(2):211-21).
(3) Yu-Jen Chang, U-Ser Jeng, Ya-Ling Chiang, Ing-Shouh Hwang, and Yun-Ru Chen*. Glycine-Alanine Dipeptide Repeat from C9orf72 Hexanucleotide Expansions Forms Toxic Amyloids Possessing Cell-to-cell Transmission Property. (2016) J Biol. Chem., 291(10):4903-11.
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TK16 Prediction of protein complex structures by GALAXY Chaok Seok Department of Chemistry, Seoul National University, Seoul 08826, Korea
Association of proteins is abundant in nature and is intimately related with the physiological functions of proteins, such as in metabolism, signal transduction, or immunity. Information on the protein complex structure is therefore important to obtain a molecular-level understanding of protein functions and their regulation. In this presentation, I will introduce the softwares and web servers (available in http://galaxy.seoklab.org) for protein complex structure prediction developed in our group such as GalaxyPPDock for ab initio docking, GalaxyPepDock for protein-peptide docking, and GalaxyHomomer for protein homo-oligomer structure prediction. We have observed that applicability and performance of the programs are improved further by combining the docking methods with template-based modelling, loop modeling, and refinement methods developed in the group. These methods have proven to be successful in blind prediction experiments such as CASP (Critical Assessment of techniques for protein Structure Prediction) and CAPRI (Critical Assessment of PRedictions of Interactions). On-going progress in incorporating experimental restraints for complex structure prediction will also be discussed.
