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Download 蛋白质结构基础(Introduction of Protein Structure)
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Molecular Biophysics 分子生物物理学 分子水平 研究生物体系物理学性质、行为 结构 功能 Molecules in Biosystem Biopolymers: Nucleic acid (DNA, RNA) Protein Saccharide Lipid Other PROTEIN STRUCTURE 1965年中国在世界上首次用化学方法 人工合成的蛋白质-牛胰岛素 S—S A链 Gly.Tle.Val.Glu.Gln.Cys.Cys.Aln.Ser.Val.Cys.Ser.Leu.Tyr.Gln.Leu.Glu.Asn.Tyr.Cys.Asn OH 6 7 1 2 20 11 S S B链 S S Phe.Val.Asn.Gln.His.Leu.Cys.Gly.Ser.His.Leu.Val.Glu.Ala.Leu.Tyr.Leu.Val.Cys.Gly.Glu.Arg. 1 2 19 7 Gly.Phe.Phe.Tyr.Thr.Pro.Lys.Ala OH 30 牛胰岛素的化学结构 Hierarchy of Protein Structure by Linderstrøm-Lang Primary Structure Secondary Structure supersecondary Structure or motif domain Tertiary Structure Quaternary Structure Property of amino acid Chiral COOH NH2 CH3 H L alanine zwitterion Uncharged structure Minor component Dipolar ion, or zwitterion Major component Classificatory of amino acid based sidechains (R groups) Non-polar Polar G,A,V,L,I; F,W, P,M, neutral S,T, N,Q acidic D,E; C,Y basic R,K, H Histidine ? Protein Primary Structure Side chain Carboxyl/C terminus Backbone Peptide bond Amine/N terminus Pauling & Corey C-N, 0.149nm C=N,0.127nm ? =180 =180 C C =0 N,C =0 Minimal Distance (Å) between nonbonding atom (G.N.Ramachandran) C O N H C O N H 3.20 (3.0) 2.80 (2.70) 2.70 (2.60) 2.90 (2.80) 2.70 (2.60) 2.70 (2.60) 2.40 (2.20) 2.40 (2.20) 2.40 (2.20) 2.00 (1.90) phi (), psi (Y), and omega (W) Relation with Energy and distance interaction Relation with Energy and distance charger-charge r -1 r -2 r -3 r -4 r -6 r -6 charger-dipole dipole-dipole charge-induced dipole dipole-induced dipole Transient dipoleinduced dipole Van der Waals force 10 kJ·mol-1,range:0.3~0.5 nm Lennard-Jones potential A B E 6 12 r r Hydrogen bond H-bond definition, H-bond location O….H-X Hydrogen bonds can vary in strength from very weak (1-2 kJ mol−1) to extremely strong (40 kJ mol−1), so strong as to be indistinguishable from a covalent bond, as in the ion HF2−. Typical values include: O—H...:N (7 kcal/mol) O—H...:O (5 kcal/mol) N—H...:N (3 kcal/mol) N—H...:O (2 kcal/mol) H O X Protein Secondary Structure 1951, Pauling p= 0.54nm P z0= 0.15nm Z0 = -57 = -47 Helices repetitive secondary structure C Helices are the most abundant form of secondary structure containing approximately 32-38% of the residues in globular proteins (Kabsch and Sander, 1983) a-helix 310 helix p-helix N Parameters of secondary structure n r P 3.613 -57 -47 3.6 0.154 0.55 310 -49 -26 3.0 0.200 0.60 p -57 -7 4.4 0.115 0.51 Paral- -119 +113 2.0 0.320 0.64 Antiparal- -139 +135 2.0 0.340 0.68 [n] is the number of residues per helical turn [r] is the helical rise per residue (nm) [p] is the helical pitch (nm). Parameters of secondary structure H-bond 3.613 i, i+4 Atoms in Hbond loop 13 radius 310 i, i+3 10 1.9 p i, i+5 16 2.8 2.3 a-helix introduction 32-38% of all residues in globular proteins The average length of an alpha helix is 10 residues. Found(-64 +/- 7, -41 +/- 7) / ideal(-57.8, -47.0) The structure repeats itself every 5.4 Å along the helix axis, i.e. we say that the a-helix has a pitch of 5.4 Å. a-helices have 3.6 amino acid residues per turn, i.e. a helix 36 amino acids long would form 10 turns. The separation of residues along the helix axis is 5.4/3.6 or 1.5 Å, i.e. the a-helix has a rise per residue of 1.5 Å Why alpha-helix is abundant in native globular protein? the phi and psi angles of the alpha helix lie in the center of an allowed, minimum energy region of the Ramachandran (phi, psi) map. the dipoles of hydrogen bonding backbone atoms are in near perfect alignment. the radius (2.3 angstrom)of the helix allows for favorable van der Waals interactions across the helical axis side chains are well staggered minimizing steric interference • CO group toward carboxyl terminus • NH group toward amide terminus • H-bond, i-(i+4) • Side chain: i-(i+3); i-(i+4) • interactions between i and i+4 stabilize helix Distortions of a-helices The majority of a-helices in globular proteins are curved or distorted somewhat compared with the standard Pauling-Corey model. Why? 1. The packing of buried helices against other secondary structure elements in the core of the protein 2. Proline residues induce distortions of around 20 degrees in the direction of the helix axis 3. Solvent. Exposed helices are often bent away from the solvent region. This is because the exposed C=O groups tend to point towards solvent to maximise their Hbonding capacity, i.e. tend to form H-bonds to solvent as well as NH groups. 310 helix introduction Only 3.4% of the residues are involved in 310 helices, and nearly all those in helical segments containing i-i+3 hydrogen bonds. Ideal (-74.0, -4.0) / found (-71.0 and -18.0) CO---HN hydrogen bond: i-i+3 Standard 310 helix Proline helix Left handed helix 3.0 residues per turn pitch = 9.4 Å No hydrogen bonding in the backbone but helix still forms. Poly-glycine also forms this type of helix Collagen: high in Gly-Pro residues has this type of helical structure p-helices introduction The pi helix is an extremely rare secondary structural element in proteins. the backbone C=O of residue i hydrogen bonds to the backbone HN of residue i+5. i- - i + 5 H-bonds 2.8angstrom 1. the phi and psi angles of the pure pi helix ( -57.1, -69.7) lie at the very edge of an allowed, minimum energy region of the Ramachandran (phi, psi) map. 2. the pi helix requires that the angle tau (N-CaC') be larger (114.9) than the standard tetrahedral angle of 109.5 degrees. 3. the large radius of the pi helix means the polypeptide backbone is no longer in van der Waals contact across the helical axis forming an axial hole too small for solvent water to fill. 4. side chains are more staggered than the ideal 3.10 helix but not as well as the alpha helix. H-bond: 1-5 Helical wheel tools alpha-helix, surface of protein, barrier amphiphilic protein design projects by Degrado, USA Helix dipole helix macrodipole The partial charges on the amide hydrogen and carbonyl oxygen are shown in units of the elementary charge contributing to an overall dipole moment of 3.46 Debye units. Sheet 20-28% (Kabsch & Sander, 1983; Creighton, 1993) a repeating secondary structure Parameters of secondary structure n r P 3.613 -57 -47 3.6 0.154 0.55 310 -49 -26 3.0 0.200 0.60 p -57 -7 4.4 0.115 0.51 Paral- -119 +113 2.0 0.320 0.64 Antiparal- -139 +135 2.0 0.340 0.68 [n] is the number of residues per helical turn [r] is the helical rise per residue (nm) [p] is the helical pitch (nm). -139 and +135 Parallel sheet Antiparallel sheet Twists about 30 degrees per residue in right-handed sense Left-handed: crossover angel Right-handed: progressive H-bond twist Parallel sheets are less twisted than anti-parallel and are always buried. Bulges One residue backbone, two H-bonds Strand connections Beta-hairpin Crossover connection: right-handed left-handed Turn 1. 2. 3. that serve to reverse the direction of the polypeptide chain Surface of the protein Antibody recognition, phosphorylation, glycosylation, hydroxylation Gamma-turn 1. H-bond: i----i+2 2. (70, -60) and (-70, 60) for i+1 residue Type I and I’ turn 1. H-bond: i----i+3 2. (-60, -30) and (-90, 0) for i+1, i+2 residues 2.3.3. Type II and II’ turn The backbone dihedral angles of residue are (-60, 120) and (80, 0) of residues i+1 and i+2, respectively of the type II turn. the hydrogen bond between CO of residue i and NH of residue i+3. This is a single turn of right-handed (III) and left-handed (III') 3.10 helix, respectively. The backbone dihedral angles of residue are (-60, -30) and (-60, -30) of residues i+1 and i+2, respectively of the classical type III turn. 2.3.4. Other structures 1. Loop random coil 2. Paperclips cap of a-helix Identification of secondary structure Identification without 3D structure CD 可信度: a-helix, 97%; sheet 75%; 50% turn, 89% other From Manavalan & Johnson, 1987 FTIR amide band I 1600-1700 NMR • coupling constant: 3JHAHN right-handed a-helix, phi = -57, 3JHAHN = 3.9 Hz right handed 3.10 helix, phi = -60, 3JHAHN = 4.2 Hz antiparallel -sheet, phi = -139, 3JHAHN = 8.9 Hz parallel -sheet, phi = -119, 3JHAHN = 9.7 Hz left-handed a-helix, phi = 57, 3JHAHN = 6.9 Hz Prediction of secondary structure (a). Homology. If sequence >25-30%, structure similarity (b). Statistical. Chou & Fasman (1978). (c). Stereochemical Schiffer and Edmundson (1967) Motif & domain 超二级结构motif 相邻的二级结构单元组合在一起, 彼此相互作用,排列形成规则的、 在空间结构上能够辨认的二级结构 组合体,并充当三级结构的构件 (block building),成为超二级 结构,介于二级结构与结构域之间 的结构层次。 常见的几种超二级结构形式 a.α-loop-α; b.β-α-β; c.β-loop-β; d. Rossmann折叠; E,f,g. 回形拓扑结构 细胞色素C α-loop-α 细胞核抗原的β-α-β结构 β-α-β 纤溶酶原的β-loop-β结构 结构域domain 多肽链在超二级结构的基础上进一 步折叠成紧密的近乎于球状的结构, 这种结构称为结构域domain 结构域的特点 • (1)结构域是球状 蛋白质的独立折叠 单位。对一些较小 的球状蛋白质分子 或亚基来说,结构 域和三级结构是一 个意思。 • 例如红氧还蛋白, 核糖核酸酶、肌红 蛋白等。 • (2)对于较大 的球状蛋白质或 亚基,其三级结 构往往由两个或 多个结构域缔合 而成也即它们是 多结构域的,例 如免疫球蛋白的 轻链含2个结构 域。 结构域有时也指功能域。功能域可以是一个 结构域,也可以是由两个结构域或两个以 上结构域组成,从功能角度看许多多结构 域的酶,其活性中心都位于结构域之间, 因为通过结构域容易构建具有特定三维排 布的活性中心。结构域之间常常只有一段 柔性的肽链连接,形成所谓铰链区,使结 构域容易发生相对运动,这是结构域的一 大特点。结构域之间的这种柔性将有利于 活性中心结合底物和施加应力。 Protein Tertiary Structure 三级结构指一个不可分的单元(分 子)的完整的三维空间结构。对于 蛋白质,此单元通常是共价连接的 一个分子。 目前已经测出三级结构的生物大分 子都储存在蛋白质数据库中 (Protein data bank,PDB),借助 软件可查阅显示其空间结构,还可 以在不同方向旋转以获得空间结构 的细节。 嗜热菌蛋白酶与人碳酸酐酶的结构图 研究蛋白质三级结构的方法 X射线晶体衍射(X-ray crystallography) 多维核磁共振( multi-dimensional NMR) 三维电子显微镜技术(3-dimensiional EM) 扫描探针显微术( Scanning Probe Microscopy ,SPM) Protein Quaternary Structure 独立的三级结构之间的非 共价缔合称为四级结构。这些 独立的三级结构称为亚基或亚 单位。