生物化学-蛋白质课件

Chapter 2 Protein（蛋白质）Amino Acids(氨基酸)A typical structure of amino acid（氨基酸）（氨基酸）氨基酸的一些重要性质(1)the capacity of polymerization （具有自身聚合的能力）(2)with acid/base properties(zwitterion；两性离子)（具有酸和碱的性质；属于两性离子）(3)20 AAs contain varied structure and chemical functionality in the amino acid side chain（支链）（20种不同的氨基酸具有不同的支链结构）(4)chirality&enantiomers（手性；对映体）（大多数氨基酸都具有光学活性）Classification of amino acids（氨基酸分类）pBasic structure:H2NCHCOOH（基本结构）a.Hydrophobic amino acids（疏水性氨基酸）b.Alanine(A)R=CH3 （丙氨酸）c.Valine(V)R=CH(CH3)2 （缬氨酸）d.Proline(P)R=C4H7N （脯氨酸）e.Phenylalanine(F)R=C6H5CH2 （苯丙氨酸）f.Methionine(M)R=CH2CH2SCH3 （苏氨酸）g.Isoleucine(I)R=CHCH3CH2CH3 （异亮氨酸）h.Leucine(L)R=CH2CH(CH3)2 （亮氨酸）i.Tryptophan(W)R=CH2C8H6N （色氨酸）Rpb.Charged amino acids（带电荷的氨基酸）pAspartic acid(D)R=CH2COOH （天冬氨酸）pGlutamic acid(G)R=CH2CH2COOH （谷氨酸）pLysine(K)R=CH2CH2CH2CH2NH2（赖氨酸）pArginine(R)R=CH2CH2CH2NHC=NH(NH2)（精氨酸）pc.Polar amino acids（极性氨基酸）pSerine(S)R=CH2OH （丝氨酸）pThreonine(T)R=CHOHCH3 （苏氨酸）pTyrosine(Y)R=CH2C6H4OH （酪氨酸）pHistidine(H)R=CH2C3H4N2 （组氨酸）Classification of amino acids(continued)pc.Polar amino acids(continued):pCysteine(C)R=CH2SH （半胱氨酸）pAsparagine(N)R=CH2CONH2 （天冬酰氨）pGlutamine(Q)R=CH2CH2CONH2（谷氨酰氨）pGlycine(G)R=H （甘氨酸）Classification of amino acids(continued)Acid-base chemistry of amino acids（氨基酸的酸碱性）（氨基酸的酸碱性）The 1st dissociation一级电离一级电离The 2nd dissociation二级电离二级电离The titration curve of glycine甘氨酸的滴定曲线甘氨酸的滴定曲线等电点等电点Isoelectric point,pI,isdefined as the pH wherea polyprotic molecule hasa net Charge of 0.氨基酸在净电荷为零时氨基酸在净电荷为零时的溶液的溶液pH值，用值，用pI表示。表示。酸性氨基酸和碱性氨基酸的滴定曲线酸性氨基酸和碱性氨基酸的滴定曲线Reactions of amino acidsThe side chain reactions（支链基团的反应）（支链基团的反应）Stereochemistry(立体化学立体化学)of amino acidsSpecific terms of stereochemistryAsymmetry（非对称）（非对称）Chiral(手性的手性的)Enantiomers(对映体对映体)Dextrorotatory(右旋右旋)Levorotatory(左旋左旋)Configurations of amino acids（氨基酸的构型：与甘油醛比较）（氨基酸的构型：与甘油醛比较）Spectroscopic properties of amino acids the ultraviolet spectra（氨基酸的紫外光谱）（氨基酸的紫外光谱）必需氨基酸 只存在食物中，动物无法合成，只能由食物中摄取，这些氨基酸被称为必需氨基酸。动物需摄取必需氨基酸以制造蛋白质。由于不同物种的化合能力不同，对于某一物种是必需氨基酸的，对另一物种则不一定是必需氨基酸。人体无法合成的氨基酸包括：人体无法合成的氨基酸包括：苯丙氨酸苯丙氨酸（Phenylalanine）缬氨酸缬氨酸（Valine）苏氨酸苏氨酸（Threonine）色氨酸色氨酸（Tryptophan）异亮氨酸异亮氨酸（Isoleucine）亮氨酸亮氨酸（Leucine）甲硫氨酸甲硫氨酸（Methionine）赖氨酸赖氨酸（Lysine）组氨酸组氨酸（Histidine）精氨酸精氨酸（婴儿婴儿）（）（Arginine）Proteins:their primary structure and biological functions（蛋白质的一级结构和生物功能）蛋白质是线性高聚物蛋白质是线性高聚物Proteins are linear polymers of amino acids Amide bondmax=212 nmPartial double-bond酰胺键两端相邻两个酰胺键两端相邻两个-碳原子碳原子处于反式位置于反式位置The peptide bond is a partial conjugated double bond Polar backbone（酰胺键具有共轭双键性质）（酰胺键具有共轭双键性质）The atoms of a amide group are coplanar（共平面）（共平面）and lying between the two alpha-C atoms酰胺键的原子处于同一平面酰胺键的原子处于同一平面Peptide classification（多肽分类）pPeptide is composed of at least two amino acid residues（氨基酸残基）connecting by peptide bond.pDipeptide,tripeptide,tetrapetide,pOligopeptide（寡肽）pPolypeptide（多肽）pProtein（蛋白质）p氨基酸残基：p二肽，三肽，四肽，寡肽，多肽，蛋白质p 氨基酸残基增加 Size of proteins（蛋白质分子的大小）Proteins are composed of one or more polypeptide chains.（蛋白（蛋白质分子由一条或者多条分子由一条或者多条肽链构成）构成）Subunit（亚基）：Monomeric（单肽链）;multimeric（多肽链）;Homomultimeric;heteromultimeric;Insulin（胰岛素）:（A链21氨基酸；氨基酸；B链30氨基酸氨基酸）Glutamate dehydrogenase（谷氨酸脱（谷氨酸脱氢酶酶）：）：6Protein sequence:from N terminal to C terminal蛋白质序列：氮端蛋白质序列：氮端-碳端碳端Ribonuclease A（核糖核酸酶（核糖核酸酶 A）Architecture of proteins蛋白质的形状蛋白质的形状Protein shapes:fibrous,globular and transmembrane proteins纤维；球型；跨膜纤维；球型；跨膜The levels of protein structure蛋白结构的分级蛋白结构的分级Primary structure（一级结构）（一级结构）Secondary structure（二级结构）（二级结构）Tertiary structure（三级结构）（三级结构）Quaternary structure（四级结构）（四级结构）2nd structures of a peptide-helex（螺旋螺旋）-sheets（折叠折叠）The tertiary structure of chymotrypsin（糜蛋白酶的三级结构）（糜蛋白酶的三级结构）The quaternary structure of hemoglobin（血红蛋白的四级结构）（血红蛋白的四级结构）Hb:22Protein conformation（蛋白质的构象）（蛋白质的构象）Conformation and configurationAnalysis and separation of proteins蛋白质的分析和分离Methods for protein assay（光度法）Lowry method Dye binding UV spectrometrySeparation method(mostly based on charges and sizes)Precipitate（沉淀）:salts Centrifuge（离心）Chromatographic methods（色谱；层析）Gel electrophoresis（凝胶电泳）：SDS-PAGEA typical scheme for separation of a protein（如何分离蛋白质）粗提物沉淀纯化物1纯化物2最后纯品盐析离子交换层析分子筛层析亲和层析比活性0.108比活性7.2比活性38.3比活性152比活性0.425比活性=总活性/蛋白质总含量Salt precipitation of globular proteins,consider the solubility of proteins in aqueous solution.蛋白质在溶液中的溶解度与溶液离子强度和蛋白质在溶液中的溶解度与溶液离子强度和pH密切相关密切相关 等电点等电点AA Analysis of Proteins（蛋白质的氨基酸分析）Acid hydrolysis of proteins（酸水解）AA analysis of proteins:auto-analyzer（用于分析蛋白质中各种氨基酸的相对含量）HPLC chromatogram（色谱图）（色谱图）of amino acidsAA compositions of proteins蛋白质中氨基酸的平均含量（%）pAla 9.0pArg 4.7pAsn 4.4pAsp 5.5pCys 2.8pGln 3.9pGlu 6.2pGly 7.5pHis 2.1pIle4.6 Leu7.5Lys7.0Met1.7Phe3.5Pro4.6Ser7.1Thr6.0Trp1.1Tyr3.5Val6.9 Determining the amino acid sequence of a protein蛋白质一级结构（序列）的测定蛋白质一级结构（序列）的测定A protein has a distinctive AA sequenceSangers work in 1953,disclosure of his study oninsulin（胰岛素）（胰岛素）sequenceProtein sequencing strategy（蛋白质测序策略）（蛋白质测序策略）Basic steps 1 7（基本步骤）（基本步骤）Protein sequencing strategy（蛋白质测序的主要步骤）pStep 1 Separation of polypeptide chains（分离多肽链）Purified form of protein（纯化蛋白质）Dissociate the composed subunits(多聚体分离)Separation of different chains（分离不同的多肽链）Step 2Cleavage of S-SBond(s)打开二硫键打开二硫键Step 3A.N-terminal analysis Edman degradation（氮端分析：（氮端分析：Edman 降解降解）Automation of Edman reaction:Edman sequenatorStep 3B.C-terminal analysis using carboxypeptidase（碳端分析：羧肽酶法）Carboxypeptidase has 4 subtypes（亚类）A,B,C and Y,each has its own effective manner.Step 4&5 Fragmentation of peptide chain by endopeptidase利用各种内切酶使肽链断裂利用各种内切酶使肽链断裂Trypsin（胰蛋白酶）（胰蛋白酶）Cleavage site:R and K on the C-side;Chymotrypsin（糜蛋白酶）（糜蛋白酶）Cleavage site:Phe,Tyr and Trp on the C-side;Other endopeptidase（其它内切酶）（其它内切酶）Lys-C:cleave at LysCyanogen bromide cleavage（利用化学方法内切）（利用化学方法内切）Selectively at Met（蛋氨酸）（蛋氨酸）Step 6 Reconstruction of the overall amino acid sequence（序列重组）（序列重组）Exemplified by catrocollastatin-CStep 7 Location of S-S bonds（二硫键定位）（二硫键定位）Diagonal electrophoresis(对角电泳对角电泳)Mass spectrometry(MS)a powerful tool for peptide sequencing质谱法测序质谱法测序Whats MS(质谱质谱)?Mass spectrometry,also called mass spectroscopy,is an instrumental approach that allows for the mass measurement（质量测定）（质量测定）of molecules.Briefly,MS is an analytical tool used for measuring the molecular weight(MW;分子量分子量)of a molecule.（质谱是一种测量物质分子量的分析工具）（质谱是一种测量物质分子量的分析工具）What Information does Mass Spectrometry Provide?质谱能够提供哪些信息？Molecular weights（分子量）:for macromolecules,e.g.proteins,MS has an accuracy of 0.01%of the total molecular weight.For small organic molecules the molecular weight can be measured to within an accuracy of 5 ppm.Structural information（结构信息）:elucidation of structure of organic compounds,and of sequences of peptide or oligonucleotide.（有机分子结构；蛋白质的氨基酸序列；核酸序列）pA mass spectrometer determines the molecular weight of chemical compounds byp（质谱分析包括以下三个环节）pIonizing（电离）pSeparating（分离）pMeasuring mass-to-charge ratio(m/z)of the molecular ions（测量质荷比）The basic operation of a MS:(1)to evaporate and ionize molecules in a vacuum,creating gas-phase ions;（蒸发并使样品电离）(2)to separate the ions in space and/or time based on their m/z ratios;（按照质/荷比分离样品离子）(3)to measure the amount of ions with specific m/z ratios.（测量）The Components of a Mass Spectrometer质谱仪结构图Inlet system Ion SourceAnalyzerIon DetectorComputerMass Spectrumm/z离子源离子源检测器检测器分析器分析器质谱图质谱图进样装置进样装置Finnigan Corporations Benchtop LASERMAT 2000Mass spectrometry for biomacromolecules:classified on the basis of ionization source根据离子源，蛋白质质谱主要分为两种ESI-MS:Electrospray Ionization Mass Spectrometry （电喷雾电离）MALDI-MS:Matrix Assisted Laser Desorption Ionization Mass Spectrometry （基质辅助激光解吸电离）Ionization methods in MS analysis of macromoleculesIon source:Electronspray ionization(ESI)电喷雾电离蛋白质的原理示意图电喷雾电离蛋白质的原理示意图ESI-MS spectra of the protein,aerolysin K蛋白质的质谱图蛋白质的质谱图MALDI:Matrix Assisted Laser Desorption Ionization基质辅助激光解吸电离方法电离蛋白质示意图基质辅助激光解吸电离方法电离蛋白质示意图Tandem MS:MS-MS串联质谱串联质谱Peptide mass fingerprinting（多肽指纹图谱）AA sequence database of proteins（蛋白质序列数据库）SWISS-PROTNCBIPDBMS/MS spectra of peptide FESNFNTQATNRNature of amino acid sequence蛋白质氨基酸序列具有的特征性Fibrous proteinsand some specific proteins have their amino acid compositions differ from that of norms.（纤维蛋白含有与其他蛋白质不同的氨基酸组成）The unique amino acid sequence of a protein reflects its own particular personality（蛋白质氨基酸序列反映了自身的结构和功能特性）Sequence similarities between proteins imply evolutionary relatedness（蛋白质之间的序列相似性隐含物种的进化关系）Homologous proteins from different organisms sharea significant degree of sequence similarity:cytochrome c细胞色素细胞色素c的序列相似性与物种的亲缘关系的序列相似性与物种的亲缘关系The phylogenetic tree for cytochrome c细胞色素细胞色素c的序列相似性的序列相似性与进化树与进化树The amino acid sequence homology of hemoglobin and myoglobin血红蛋白（血红蛋白（Hb）和肌红蛋白（）和肌红蛋白（Mb）的进化关系）的进化关系Biological functions and classification of proteins蛋白质按功能分类蛋白质按功能分类1.Enzymes 酶酶2.Regulatory proteins（调节蛋白）（调节蛋白）They do not perform any chemical transformation but can regulate the abilities of other proteins3.Transport proteins（转运蛋白）（转运蛋白）They carry and transport substancesfrom one site to other site.4.Storage proteins（存储蛋白）Provide a reservoir of an essential nutrient.5.Contractile（收缩）and motile（游动）proteinsTo endow cells with unique movement,such as cell division,muscle contraction and cell motility.6.Structural proteins（结构蛋白）To provide strength and protection to cells and tissues,creating and maintaining special biological structure of organisms.7.Scaffold proteins(adaptor proteins；支架蛋白；支架蛋白)Having a structure of organized modules which can recognize and binding other proteins,and the resulting assemblage can perform its special biological functions.8.Protective and exploitive（防护与进攻）proteinsIn contrast to the structural proteins,these protein have active role in cell defense and protection.9.Exotic proteins（异常蛋白）can display rather impressive can rare properties.10.Conjugated proteins（结合蛋白）contains other chemical groups(non-amino acid)as an integral part of their structure.They are classified according to the chemical nature of the non-amino acid groups.The structure of protoporphyrin and heme原卟啉原卟啉血红素血红素蛋白质的高级结构Secondary,tertiary and quaternary structureThe function of a protein is determined by its 3-dimensional shape or its conformation.(蛋白质的功能决定于其三维结构和构象)The non-covalent forces maintaining protein structure include hydrogen bonds,hydrophobic interactions,electrostatic bonds and Van de Waals forces.(几种非共价作用力维持蛋白质的正确构象：氢键；疏水作用；静电作用和范氏引力)Hydrogen bonds（氢键）it is the main force to maintain the secondary structures of a protein.Hydrophobic interactions（疏水作用）formed by nonpolar side chains of amino acid residues,preferably clustering in the hydrophobic core of a protein.Van der Waals interactions（范氏作用力）are ubiquitous in proteins p维持蛋白质三维结构和构象的主要作用力Electrostatic interactions（静电作用）（静电作用）backboneSide chainsAmino acid sequence of a protein is highly informative,which contains all the information for folding the polypeptide chain into its native structure.（蛋白质如何折叠成正确的三维结构由基因编码的氨基酸序列所决定）By using database of protein structures,people are able to predict the 3d structure of a polypeptide according to its primary sequence(利用已有的序列-结构数据库，可以根据氨基酸序列预测多肽的三维结构).Secondary structure in protein蛋白质的二级结构蛋白质的二级结构Amide planes:the basic structural blocks of protein（酰胺平面是蛋白质高级结构的（酰胺平面是蛋白质高级结构的基本模块）基本模块）Rotations:angles,（酰胺平面的旋转酰胺平面的旋转）A hydrogen bond between the amide proton and carboxyloxygen of adjacent peptide groups.（维持蛋白质二级结构的主要作用力来自于氢键）（维持蛋白质二级结构的主要作用力来自于氢键）It is the H bonds that link one peptide group to another,toconstitute secondary structure for proteins:-helices（-螺螺旋）旋）and-pleated sheets（-折叠）折叠）The alpha-helix-螺旋螺旋Alpha-helices presented as coiled ribbon drawings-螺旋的一种表示方式螺旋的一种表示方式Loops Alpha-helix has a net dipole moment(-螺旋具有偶极矩螺旋具有偶极矩)Some AA prefers to form alpha-helix(一些氨基酸易于形成一些氨基酸易于形成-螺旋螺旋)pleated sheets：折叠折叠 Parallel（平行）（平行）Antiparallel（反平行）（反平行）Hydrogen bonds interstrand维系维系-折叠折叠结构的是构的是链间氢键其他二级结构其他二级结构 turns：转角转角Gly and Pro两种氨基酸在转角结构中常见两种氨基酸在转角结构中常见Prediction of structure of helices by hydropathy plotting the amino acid sequence of a protein根据氨基酸序列的疏水性分布，根据氨基酸序列的疏水性分布，预测-螺旋螺旋Hydrophobicity Scales氨基酸的疏水性数值pPhe MetIleLeuValCysTrpp3.7 3.43.12.82.62.01.9pAla ThrGlySerProTyrHisp1.6 1.21.00.6-0.2-0.7-3.0pGln AsnGluLysAspArgp-4.1-4.8-8.2-8.8-9.2-12.3 Motif(模体模体):simple combinations of a few secondary structure elements with a specific arrangements.（多个二级结构可以组合成（多个二级结构可以组合成超二级结构超二级结构）These motifs are also called supersecondary structure(超二级结构超二级结构).Topology diagram(拓扑图拓扑图):a simplified schematic representation of the secondary structure（二级结构的简化表示方法）（二级结构的简化表示方法）(a)The path of main chain composed of a-motif;(b)Topological diagram of the structure in(a).Loop area蛋白质的活性部位通常在蛋白质的活性部位通常在loop区域区域substrateThe active site is usually located in loop areaThe active site of arabinose-binding protein of E.ColiActive siteSome special parallel-sheet proteinsMolecular Chaperone分子伴侣分子伴侣蛋白质在其多肽链合成出来后，蛋白质在其多肽链合成出来后，需要其他分子，即分子伴侣将需要其他分子，即分子伴侣将其折叠为正确的三维结构其折叠为正确的三维结构Proteins that help fold globular proteinsQuaternary structure of protein:subunits interactions蛋白质的四级结构：亚基间的作用方式蛋白质的四级结构：亚基间的作用方式The symmetry of quaternary structure蛋白质四级结构具有一定的对称方式蛋白质四级结构具有一定的对称方式