1、Biochemistry,For Specialty of Biotechnology or Bioengineering at CSUMr. XIA Jinlan,Chapter 5 Proteins: Their Biological Functions and Primary Structure 蛋白质的生物学功能和一级结构,Basic constituents and structures of Proteins 蛋白质的基本组成和结构 Architecture of protein molecules蛋白质分子的构架 Classification of Proteins 蛋白质的分类
2、 The many Biological functions of proteins 蛋白质的许多生物学功能 Some proteins have chemical groups other than amino acids 有些蛋白质含有非氨基酸基团 Protein purification 蛋白质纯化 Protein sequencing 蛋白质测序,Basic constituents and structures of Proteins 蛋白质的基本组成和结构,Proteins are linear polymers of amino acids蛋白质是线性氨基酸聚合物 Peptide
3、 bond 肽键 Peptide unit 肽单元 Proteins have four levels of basic structures蛋白质具有四级基本结构 Primary structure 一级结构 Secondary structure 二级结构 Tertiary structure 三级结构 Quaternary structure 四级结构,Peptide bond 肽键,Formation of peptide bond is by condensation of a-amino group + a-carboxyl group 肽键是由一个氨基酸的氨基和另一氨基酸的羧基缩
4、合而成 A protein is a linear sequence of amino acids linked together by peptide bonds. 系列肽键相连组成蛋白质的线性序列 Peptide bond has a resonance structure肽键具有一共轭结构,Formation of peptide bond: a-amino group + a-carboxyl group,Peptide bond 肽键 Amide linkage酰胺键,A protein is a linear sequence of amino acids linked toget
5、her by peptide bonds.,Peptide bond has a resonance structure,Protein structure,Peptide units and peptides肽单元和肽,Peptide units are planar and rigid 肽单元是平面和刚性的 Peptide is named after the number of the amino acids (AAs) residues in the peptide chain 肽的命名（或划分）是根据肽链中氨基酸残基的数量 Peptide chains written form 肽链
6、的书写方式: +H3N-glycine-threonine-phenylalanine-coo- Or simply: Gly-Thr-Phe, or G-T-F,Peptide units are planar and rigid,Classification of Peptides肽的分类,Dipeptides 双肽 With two AAs residues Oligopeptide 寡肽: 12AAs 20 AAs residues (some books: 24AAs residues) Proteins are composed of one or more polypeptide
7、 chains 蛋白质是由一条或多条多肽链组成的,Protein structure,There four levels of structure in proteins蛋白质有四级结构,Primary 一级 Linear sequence of amino acids, including the disulfide bonds 线性氨基酸序列 (包括双硫键在内) Secondary 二级 a-helix 螺旋, b-pleated sheet 折叠(片) Tertiary 三级 Folded three-dimensional structure 折叠的三维结构 Only one poly
8、peptide chain 只有一条多肽链 Quaternary 四级 Folded three-dimensional structure折叠的三维结构 Two or more than two polypeptide chains (subunits 亚基)含有两个或两个以上的多肽链,AA sequence of Bovine pancreatic Ribonuclease A,Primary structure 一级结构,“Shorthand” a-helix,“Shorthand” b-strand,S E C O N D A R YS T R U T U R E,Tertiary s
9、tructure,Myoglobin 肌球蛋白,1 subunit,Heme 血红素 一种辅基(prothetic Group),Quaternary structure,Hemoglobin 血红球蛋白,2 b-chains + 2 a-chains,Comparison between tertiary structure And Quaternary structure,Myoglobin 肌球蛋白,Hemoglobin 血红球蛋白,An example for showing the relationship among the for levels of structures,Fou
10、r levels(a) Primary (b) Secondary (c) Tertiary (d) Quaternary,Protein structure,Classification of Proteins 蛋白质的分类,Classified by functions按照功能分类 Classified by conjugated constituents按照缀合组成分类,Biological functions of proteins 蛋白质的生物学功能,Enzyme 酶 Regulatory 调节 Transport 转运 Storage 储存 Contractile and moti
11、le 收缩和运动 Structural 结构 Scaffold 骨骼 Protective and exploitive 保护性和防卫性 Exotic 奇异,See table 5.3, p121,Some proteins have chemical groups other than amino acids 有些蛋白质含有非氨基酸基团,Examples: Glycoproteins 糖蛋白 Lipoproteins 脂蛋白 Nucleoproteins 核蛋白 Phosphoproteins 磷蛋白 Metalloproteins 金属蛋白 Hemoproteins 血蛋白 Flavopr
12、oteins 黄素蛋白,See table 5.4, p127,Heme structure in hemoproteins 血红蛋白中血红素结构,Heme: one kind of prosthetic group (辅基） Protoporphyrin IX + Fe2+ 原卟啉四环+亚铁离子,Typical examples of proteins and their functions,Two oxygen-binding proteins found in vertebrates (脊椎动物中的两种氧结合蛋白) Hemoglobin 血红蛋白 Be in blood within e
13、rythrocytes, Carries O2 in the blood from the lungs to the other tissues. Myoglobin 肌红蛋白 Be in tissues of skeletal and cardiac muscle (骨骼和心肌组织中) where it stores O2.,4O2,4O2,Venous circulation,Arterial circulation,静脉循环,动脉循环,Hemoglobin (Hb),Hb(O2)4,Hb(O2)4,Hemoglobin (Hb),Physiological functions of th
14、e hemoglobin in different tissues,Tissue组织,Lung 肺,Heart,Questions: What are the basic principles the protein purification based on? What are the main techniques for separation of proteins? Throughout the purification procedure, how do you prevent the protein of interest from being denatured either b
15、y physical or biological factors? What are the techniques used to assay the protein of interest? Terms explanations: Homogenization Salting out effect Dialysis Something required to know: Semi-permeable membrane: cellophane (cellulose acetate); It is often used for separation of salt in protein solu
16、tion by dialysis.,Purification of protein Mixtures,General Principles for Bio-Separation methods 生物分离方法的基本原理,Methods based on size 基于物质大小 Size exclusion chromatography 分子筛色谱（凝胶过滤色谱 Ultra-filtration 超过滤 Ultracentrifugation 超速离心 Methods based on electric charge 基于电荷性质 Ion exchange chromatography 离子交换色
17、谱 Electrophoresis 电泳 Solubility 溶解 （区分带电与不带电所致水溶解性） Methods based on specific affinity 基于特异亲和性 Immunoaffinity chromatography 免疫亲和色谱 Immunoaffinity precipitation 免疫亲和沉降,Basic methods for separation and purification of proteins 蛋白质分离纯化的基本方法,Crude separation 粗分离 Precipitation at pI 等电点沉降 Salting in/sal
18、ting out 盐溶/盐析 Fractionation by centrifugation 沉降分级 Fine purification 精细纯化 Chromatography 色谱分离分析 Electrophoresis 电泳,Comparison in some parameters among some fractionation methods for purification of proteins 蛋白质纯化分级方法的参数比较,The fractionation scheme 分级方案,表观活性,回收百分率,Common methods for determination of
19、proteins蛋白质浓度测定的方法,UV 紫外（测定法） Lowry procedure 劳里（程序）法 Bicinchoninic acid (BCA) method 双弱金鸡纳酸法 Bradford assay 布勒佛（测试）法,The Monitoring of Proteins 蛋白质的监测,Please compare the differences in principles, sensitivity and selectivity of the four common methods for determination of proteins. 请比较四种蛋白质测定方法在原理、
20、灵敏度、选择性（抗干扰性）等方面的区别 Choice of standard protein is essential to assure the accuracy of determination. How do you choose a suitable standard protein, when you determine IgG and serum, respectively? 标准蛋白质的选择对于测定的准确度很重要。当你分别测定免疫球蛋白和血清蛋白质时，你如何正确选择标准蛋白质？,Homework:,Protein sequencing 蛋白质测序,Definition of pr
21、otein sequencing蛋白质测序的定义 The procedure to uncover the sequence of amino acids residues arranging in the protein of interest 测定目标蛋白质中氨基酸残基的排列顺序 Note 注意点 : Protein sequence is not composition expression 蛋白质序列不等于蛋白质组成 Basic strategies of protein sequencing 蛋白质测序的基本策略,Example: Sequence 序列:Val-Phe-Asp-Ly
22、s-Gly-Phe-Val-Glu-Arg Composition expression 组成表述: (Arg, Asp, Glu, Gly, Leu, Lys, Phe2, Val2),Protein sequence is not composition expression 蛋白质序列不等于蛋白质组成的表述,Protein sequencing strategy 蛋白质测序策略,Separation of polypeptide chains to get subunits (if 1subunits) 将多亚基分解成单个亚基 (如果蛋白质多于一个亚基） Cleavage of disu
23、lfide bridges to make protein linear 打开双硫键得到线性蛋白 Analysis of amino acid composition 氨基酸组成分析 Identification of the N- or C-terminal residues 末端分析 Fragmentation of the polypeptide chain 肽链肢解 Sequence determination of the oligopeptides by MS 质谱法测定寡肽链氨基酸序列 Reconstruction of the overall amino acid sequen
24、ce Location of disulfide cross-bridges,Protein sequencing 蛋白质测序,Separation of polypeptide chains to get subunits 多肽链的分离,e.g., for insulin,Cleavage/separation of subunits,Protein sequencing 蛋白质测序,Step 1,Cleavage of disulfide bonds to make protein linear 打开双硫键得到线性蛋白,Oxidation by performic acid 过甲酸氧化 R
25、eduction of S-S-, and protection of SH 双硫键还原和巯基的保护 Reductant 还原剂: 2-mecaptoethanol or dithiothreitol 2-巯基乙醇 或 硫代苏糖醇 Protectant (巯基)保护剂: Iodoacetic acid or 3-Bromopropyl-amine 碘代乙酸 或 3-溴丙胺,Protein sequencing 蛋白质测序,Oxidation by performic acid 过甲酸氧化,Protection of SH 巯基的保护,Reduction of S-S-, and protect
26、ion of SH 双硫键还原和巯基的保护,Composition analysis 组成分析,Normal procedure Hydrolysis Ion exchange chromatography Detecting agent ninhydrin (茚三酮) (commonly used) DNFB =Sanger agent DNS-Cl =dansyl chloride,Protein sequencing 蛋白质测序,Step 3,(DNFB, Sanger agent),N-terminal analysis By way of Edman degradation ( Ed
27、mans reagent )利用艾得曼降解法分析N-末端 C-terminal analysis By way of enzymatic analysis with carboxypeptidase A, B, C or Y 利用羧肽酶A，B，C或Y酶解分析C-末端,Protein sequencing 蛋白质测序,Step 4,Terminal analysis 末端分析,Edmans reagent for N-Terminal analysis N末端分析用Edman试剂,Phenylisothiocyanate 苯基异硫氰酸,Principle of N-Terminal analys
28、is N末端分析原理,Edman reagent 艾得曼试剂,Use specific carboxypeptidase A, B, C or Y to hydrolyze C-terminal peptide bond利用特异性羧肽酶水解C-末端肽键 A: not for Pro, Arg, and Lys B: only for Arg or Lys C and Y: for any terminal amino acid peptide bond Separation procedure is similar to that of N-terminal analysis （酶解后碎片）分
29、离程序类似于N-末端分析,C-Terminal analysis C末端分析,Chemically 化学法 Cyanogen bromide (CNBr) cleaves C-terminal side of Met residues 溴化氰裂解Met的C-端 Enzymatically 酶法（much more often 较常用） Trypsin cleaves C-terminal side of basic (Arg, Lys) 胰蛋白酶裂解碱性氨基酸Arg、Lys 的C-端 Chymotrypsin cleaves C-terminal side of aromatic (Phe,
30、Trp, Tyr) 胰凝乳蛋白酶裂解芳香氨基酸Phe, Trp, Tyr的C-端,Fragmentation of polypeptide chain多肽链肢解,Protein sequencing 蛋白质测序,Step 5,Please note Table 5.6, p137,Chemical cleaving 化学裂解 Cyanogen bromide (CNBr) cleaves C-terminal side of Met residues 溴化氰裂解Met的C-端,Principle of cleavage of C-terminal side of Met residues by
31、 cyanogen bromide (CNBr) 溴化氰裂解Met的C-端的原理,Over reaction总反应,Enzymatic cleaving 酶法裂解,Chymotrypsin : cleaves C-terminal side of aromatic (Phe, Trp, Tyr),Trypsin: cleaves C-terminal side of basic (Arg, Lys),Protein sequencing 蛋白质测序,Sequence determination of the oligopeptides by Mass spectrometry (MS) 质谱测
32、序,Step 6,Reconstruction of the overall amino acid sequence 所有氨基酸序列重组,By overlapping fragments to analyze the sequence of a peptide 通过寡肽片段叠加分析肽的序列,Protein sequencing 蛋白质测序,Step 7,Information derived from protein sequences,To know the similarity among proteins Evolutionary relationships Gene duplicati
33、on Post-translation processing Antibodies DNA probes,Protein sequencing 蛋白质测序,Homework:,What is Edman reagent? 什么是Edman试剂？ Please point out the strategy for composition analysis of amino acids residues of a protein.请说出蛋白质氨基酸组成分析的策略。 Please describe the basic strategy for sequencing protein. 请描绘蛋白质测序
34、的基本策略。 What amino acids C-terminal is cleaved by cyanogen bromide? 什么氨基酸的端被溴化氰裂解？ Which amino acidss C-terminal are cleaved respectively by trypsin and chymotrypsin? 胰蛋白酶和胰凝乳蛋白酶分别裂解什么氨基酸的端？ Problems: 1, 2, 4, 5 (p151) Self-study for Problem 10 (p152),Appendix 1: Methods for Purification of Proteins,
35、A minimum in solubility occurs at the isoelectric point (PI),Protein purification,Solubility of b-lactoglobulin (乳球蛋白质）as a function of pH pI=5.2,M=mol.L-1,By use of precipitation at PI,Salting out/salting in 盐析/盐溶,Solubility of horse Hemoglobin (血红蛋白）in different salt solutions,Salting in (solubliz
36、ing protein) A moderate amount of salt required to be added. Salting out (precipitation of proteins) So much salts added that certain salts compete more favorably for solvent and decrease the solubility of the protein,Protein purification,Centrifugation/ultracentrifugation,Usefulness For separation,
37、 determination of gross size and shape Principle,Protein purification,Centrifugation determined by Sedimentation rate,dx/dt is the rate at which the particle travels at distance x from the center of rotation w2x is the angular acceleration,Protein purification,Centrifugation: principle,Sedimentation
38、 rate is proportional to the molecular weight,Centrifugation: principle,Protein purification,Sedimentation constant can be estimated approximately by linear gradients of sucrose or glycerol.,Centrifugation: for determination of gross size and shape,This is not a rigorous relationship and the approxi
39、mation is best for spherical molecules.,Protein purification,The sedimentation constant is usually given by Svedberg units (S) where S=10-13 sec.,Examples,Chromatography of proteins (蛋白质色谱),Principle,Protein purification,Gel filtration or size exclusion chromatography,Principle: Based on size and sh
40、ape of molecules Small molecules enters the pores in the beads whereas larger or more elongated molecules cannot. Large or more elongated molecules are eluted first The often used porous bead: Highly hydrated, with pores of different size Polymer: polyacrylamide (Bio-Gel) Carbohydrates: Dextran 葡聚糖(
41、Sephadex) Agarose 琼脂糖 (Sepharose) Eluent Buffer （缓冲液） Application De-salt a protein mixture Estimation of the molecular mass of a protein,Purification /Chromatography of protein,Gel filtration chromatography,Schematic diagram of GFC (b) Elution curve (c) Unknown prediction,Purification /Chromatograp
42、hy of protein,Ion exchange chromatography (IEC),Principle: Based on net charge of molecules interest Anionic proteins阴离子蛋白质 Anion exchange chromatography (AEC) Cationic proteins阳离子蛋白质 Cation exchange chromatography (CEC) Beads For AEC DEAE-cellulose (纤维素) or DEAE-sephadex(葡聚糖凝胶 ) For CEC CM-cellulos
43、e or CM-sephadex Eluent: Buffer （缓冲液） Gradient NaCl （梯度NaCl溶液）,DEAE: diethylaminoethyl 二乙基氨基乙基 CM：carboxymethyl 羧（基）甲基,Purification /Chromatography of protein,Schematic diagram of IEC (b) Elution curve,Affinity chromatography,Principle Based on the specific, high affinity, noncovalent binding of a p
44、rotein to its ligand（亲和色谱） Bead: an inert and porous matrix (e.g., Sepharose) immobilized ligand Eluent: Soluble ligand (binding to protein interest) Application: an inhibitor to purify an enzyme; an antibody to purify its antigen, a hormone (e.g., insulin胰岛素) to purify its receptor, a lectin 外源凝集素
45、(e.g., concanavalin A) to purify a glycoprotein (糖蛋白),Purification /Chromatography of protein,Schematic diagram of AC (b) Elution curve,Electrophoretic methods are the best way to analyze mixtures Native polyacrylamide gel electrophoresis （Native-PAGE) 非变性聚丙烯酰胺凝胶电泳 Based on net negative charge and s
46、ize SDS-PAGE （ SDS: sodium dodecyl sulfate 十二烷基磺酸钠） Based on molecular mass or size Isoelectric focusing 等电聚焦 （pI),Electrophoresis of proteins (蛋白质的电泳),Native PAGE 非变性聚丙烯酰胺凝胶电泳,Native PAGE is the basic electrophoresis 非变性PAGE 是基本的电泳方法 Principle Based on net negative charge and size of proteins PAGel
47、 (polyacrylamide gel) As a molecular sieve (分子筛）(Pore size depends on concentrations of acrylamide and the cross-linking reagent, methylene bisacrylamide) Small molecules pass readily Larger molecules are retarded Migration of protein in the Electric field The greater the net negative charge, the fu
48、rther the protein will migrate,Purification/ Electrophoresis of proteins,Electrophoresis direction cathode to anode (top to bottom) Bromophenol blue is added in the sample Indicating the progress of Elelctrophoresis Buffer pH 9 (?) Electric voltage 300V Time: 30-90min,Native PAGE 非变性聚丙烯酰胺凝胶电泳,Purifi
49、cation/ Electrophoresis of proteins,SDS-PAGE,Based only on their mass, because The proteins are denatured and coated with an overall negative charge, i.e., there no difference in charge Denatured agent: Reducing agent: break all the disulfide bonds 2-mecaptoethanol (2-巯基乙醇) dithiothreitol （DDT, 二硫代苏糖醇） Strong anionic detergent: disrupts nearly all the noncovalent interactions in the protein, unfolding the polypeptide chain SDS Application in determination of protein sample Purity Molecular mass (within 2% error) Number of polypeptide subunits,
