研究生院_膜蛋白seminar2013-12-9-柳振峰

EM structure of AQP-0 at 1.9 Å resolution
Hite, R. K., Raunser, S. and Walz, T. (2007) Curr. Opin. Struct. Biol. Gonen T., et al. (2005) Nature (2005)
冷冻电镜单颗粒三维重构方法
• 1.4 % of all structures in PDB belong to membrane proteins
(Only 407 unique MP structures)
膜蛋白结构研究的重要意义和新颖性（The gold mine); 膜蛋白的 挑战性 ( Challenge ); 机会与风险并存的一个前沿领域。
膜蛋白有何特殊性质，为什么难以纯化?
Surface presentation of LHCII (Hydrophobic region in gray)
Oil and water mix phase separation
去污剂的特性: 微囊化（micellization）
Anatrace: Detergent and their use in membrane protein sciences. 2011
膜蛋白结构生物学研究进展
柳振峰
中国科学院生物物理研究所 2013-12-9 liuzf@sun5.ibp.ac.cn/64881481
提
纲
Part I: 研究背景与方法学介绍 Part II: 研究实例: LHC-II 和 MscL的结构与功能研究
Part I:
研究背景与方法学介绍
General Background Introduction and Methodology
狂野的西部（The wild west）
“Wild West often refers to the American frontier period of the Western United States in the 19th century.” “Most often, the term "American West" is used for the area west of the Mississippi River during the 19th century.” ------ Wikipedia
1988 Nobel Prize
in Chemistry
Nobel Prizes in Chemistry, 2003 and 2012
High resolution structure of K+ channel Structure of 2-AR in complex with Gs Brian Kobilka, Nobel Lecture, 2012 Roderick Mackinnon, Nobel Lecture, 2003
2-adrenergic receptor in complex with G proteins (PDB: 3SN6)
• 细菌和癌Байду номын сангаас细胞的多药抗性（MDR转运蛋白）
膜蛋白: 结构生物学的“狂野西部”
Membrane Proteins accounts for • 20-30% all ORF in prokaryotic and eukaryotic genomes
中国的美丽西部
周克义 摄
First X-ray Structure of Membrane Protein
Cytc
Deisenhofer, J.
M
L
Huber, R.
Michel, H.
H
Photosynthetic Reaction Center from R. viridis
Deisenhofer, J. Epp O., Miki, K., Huber R. and Michel H. (1984) J. Mol. Biol. 180: 385-398 Deisenhofer, J. Epp O., Miki, K., Huber R. and Michel H. (1985) Nature 318: 618-624 Deisenhofer J., Michel, H. and Huber, R. (1989) EMBO J. 8:2149-2170
Small amphiphiles 概念
Michel H. Crystallization of membrane proteins. (1983) TIBS 8: 56-59.
http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
Membrane protein structure consortiums
http://www.mpsi.ac.uk/
Membrane protein structure consortiums
生物膜与膜蛋白
Integral polytopic MP (Transmembrane proteins)
monotopic MP
Classification of membrane proteins
Peripheral MP
膜蛋白研究的意义何在?
膜蛋白的主要功能:
信号转导 物质转运（选择性通透） 能量代谢（光合/呼吸作用） 脂类合成等酶促反应
Newby, Z. E. et al. 2011 Nature protocols 4: 619-637.
膜蛋白晶体优化的目标：获得衍射质量好的晶体 1. Small amphiphiles, 2nd detergent and other additives 2. Lipid: lipidic cubic phase or bicelle crystallization 3. Protein modification: mutation, trim, fusion or antibody fragment 4. Funnel approach
结构生物学研究实践中如何对付膜蛋白?
• 实际问题: 如何获得足够量的纯化样品？ 1. 来源: 天然或异源表达 2. 分离纯化：去污剂+传统生物化学中的蛋白分离纯化方法 3. 膜蛋白晶体生长：For studies through X-ray crystallography
天然来源膜蛋白
• Respiratory complexes: complex I, II, III and IV, rhodopsin GPCR
如何研究膜蛋白的三维结构？
• X-射线晶体学
• 电子显微学（电子晶体学和单颗粒分析）
• 多维核磁共振(NMR)方法和顺磁共振(EPR)方法
电子晶体学方法（Electron crystallography）研究膜蛋白
7 Å EM Structure of Bacteriorhodopsin
Henderson, R. and Unwin, P. N. T. (1975) Nature
去污剂对膜蛋白的增溶和抽提
Newby, Z. E. et al. 2011 Nature protocols 4: 619-637.
膜蛋白的分离纯化
• • • • Centrifugation & fractionation Affinity chromatography: IMAC et al. Ion exchange chromatography Size exclusion chromatography
Crystals of SaMscL-Cdelta26
如何评估初始搜索结果?
• 假阳性结果: 1. spherulites 2. noodles 3. salt crystals 4. lipid crystals • Diffraction tells
Spherulites under polarized lens (possible detergent crystal, usually with CnM detergentl) Photo taken by 尹妍
3.4 Å分辨率的TRPV1离子通道三维结构
Liao M. F. et al. (2013) Nature, 2013
NMR方法研究膜蛋白结构
Structure of Mitochondria UCP p7 channel from hepatitis C virus
Berardi, et al. (2011) Nature Yang, B. O. (2013) Nature
膜蛋白的分离纯化
• 去污剂（Detergent） • 纯化方法（Purification Method） • 去污剂置换（Detergent exchange） (on Ni-column, ion-exchange column, gel filtration or dialysis) • 膜蛋白浓缩（Protein concentration） • 综合因素（Concerns: excess detergent, protein purity and stability balance）
膜蛋白与人类健康和疾病相关
• GPCR, 目前市场上约 40% 的处方药物是以GPCR作为靶蛋白 （例如治过敏性哮喘的2-AR受体激动剂）； • 电压门控离子通道 (hERG, Nav1.5, Cav1.2) 与长QT综合症
Nomal QT interval
Prolonged QT interval
•Photosynthetic complexes: photosystem I and II, LHCII
•P-type ATPases: Ca2+ -ATPase
异源过表达 (Heterologous expression)
•E. coli: Prokaryotic membrane proteins: KcsA, MscL, ABC transporters, LacY/Glpt, LeuT et al. •Yeast: Eukaryotic Kv, Kir and CLC transporter (Rod Mackinnon lab), Ca2+-ATPase, Histamine H1 receptor (GPCR) •Baculovirus-Insect cell: AMPA glutamate receptor, acid sensing channel ASICs, ATP-gated P2X4 ion channel (all from Eric Gouaux lab), GPCR •Cell-free expression: EmrD, Acetabularia rhodopsin II (ARII)
Important: 1. add at least 2-10 CMC detergent in all solution used in purification to prevent MP from precipitating out 2. over-purification might destabilize MP and abolish crystallizability
http://www.nycomps.org/index.html
IBP,CAS膜蛋白结构-功能研究团队
江涛
张凯
常文瑞
柳振峰 黄亿华
2012年7月20日大分子实验室夏季PI会，王强摄
清华大学施一公&颜宁团队
Outward facing FucP structure, Nature 2010
Structure of ECF A-A’-T-S complex, Nature 2013
进入结晶前的膜蛋白质量检验：单分散性

Newby, Z. E. et al. 2011 Nature protocols 4: 619-637.
Different profiles of MP size exclusion chromatography
膜蛋白结晶
• • • 初始搜索（Initial Screen） 优化策略（Grid Screen） 三种不同类型的膜蛋白晶体