蛋白质结构与功能.pdf

Protein Structure 蛋白质结构与功能 Lu Linrong 鲁林荣 PhD Laboratory of Immune Regulation Institute of Immunology Zhejiang University School of Medicine Medical Research Building B815 819 Email Lu Linrong gmail comEmail Lu Linrong Website OutlineOutline Amino acids properties Protein structure primary secondary tertiary and quaternary structuresprimary secondary tertiary and quaternary structures methods for protein separation and purification methods to study protein structuresmethods to study protein structures Amino Acids and Peptides Amino Acids 氨基酸 Building blocks 氨基酸 g There are over 300 naturally y amino acids on earth Only 20 amino acidsOnly 20 amino acids participate in proteins formationformation Different side chains R determin the properties ofdetermin the properties of 20 amino acids 2020 Amino acidsAmino acids20 20 Amino acidsAmino acids Glycine G Leucine L Isoleucine I Valine V Alanine A Glycine G Leucine L Isoleucine I Valine V Alanine A Methionine M Asparagine N Tryptophan W Phenylalanine F Proline P Threonine T Serine S Glutamine Q Cysteine C Tyrosine Y Glutamic acid E Asparatic acid D Histidine H Lysine K Arginine R White Hydrophobic Green Hydrophilic Red Acidic Blue Basic Classifications of amino acids 1 Nonpolar hydrophobic 非极性 l h dh l 极性中性2 Polar hydrophilic 极性中性 3 Acidic 酸性3 Acidic 酸性 4 Basic 碱性 1 Nonpolar hydrophobic AAs 2 P lhd AA2 Polar uncharged AAs 3 Acidic AAs3 Acidic AAs 4 Basic AAs Special AAsSpecial AAs Glycine side chain is H very flexible Proline has two covalent bonds with backbone CH 2 CH 2 Cysteine can form disfulfide bridge 2 CHCO O N H 2 CH CH 2 2 CHCO O N H 2 CH CH 2 CH 2 CH 2 Properties of Amino AcidsProperties of Amino Acids A iid hbhidd bi 两性解离 Amino acids have both acid and base properties 两性解离 Aromatic amino acids absorb light in the near ultraviolet 近 紫外吸收 Coloured by ninhydrin 茚三酮反应 Coloured by ninhydrin 茚三酮反应 Sanger s reaction Edman reaction Amino acids have both acid and base properties 两性解离 pH pIpHpI pI isoelectric point 等电点 Th ilt ii t i thH thi htilllThe isoelectric point is the pH at which a particular molecule or surface carries no net electrical charge Aromatic amino acids absorb light in the near ultraviolet 近紫外吸收 Aromatic amino acids 芳香族氨基酸 Trp Tyr Phey Near ultraviolet Can be used for protein Can be used for protein quantitation Ninhydrin reaction 茚三酮反应 Ninhydrin reaction 茚三酮反应 440 nm for proline hydroxyproline540 nm for other AAsp yyp 羟基脯氨酸 540 nm for other AAs 黄色紫色 离子交换层析柱离子交换层析柱 茚三酮茚三酮 Sanger s reaction FDNB DNFB Determining the N termial amino acid in a polypeptide chain N 端测定 二硝基氟苯 硝基氟苯 DNP 蛋白DNP 蛋白 DNP 氨基酸氨基酸 Edman reaction PITC 苯异硫氰酸酯 1 Peptide is coupled to phenylisothiocyanate PITC The free N terminal amino group reacts i h hbf hwith the carbon of the isothiocyanate group to give the phenylthiocarbamyl PTC ditif thtidderivative of the peptide 2 Cleavage of the PTC derivative using anhydrous trifluoroacetic acid 无水三氯乙酸 to give theacid 无水三氯乙酸 to give the anilinothiozolinone ATZ derivative of the N terminal amino acid and the peptide with oneacid and the peptide with one fewer amino acid 3 The ATZ residue is converted to the phenylthiohydantoin PTH the phenylthiohydantoin PTH amino acid First the ATZ derivative is hydrolyzed under aqueous acidic conditions to give q g the PTC amino acid The acid then cyclizes to give the stable PTH derivative Protein Sequencing Protein is attached to a solid support such as a chemically modified glass diskoraporouspolyvinylidene fluoridemembraneinthereaction cartridge The machine performs the couplingcleavageconversion andcoupling cleavage conversion and identificationsteps derivatives injected into an HPLC column where its retention time is compared with thatofknownPTHaminoacid standards foreachaminoacid residue The protein may then be identified by searching a database toidentified by searching a database to match the sequence and properties of the protein Protein Sequencing Peptides 肽 Peptides 肽 Peptides are short polypeptides chains up to length of about 50 amino acids Proteins are made up of one or more polypeptides with more than 50 amino acids H OR H H2NC C H R1 OH O N C H H H R COOH H2O H2N CC H R1 O NC H R2 COOH H H O H R2H O R2 The Peptide Plane 肽平面The Peptide Plane 肽平面 The atoms of the group O C N H together with the two C H together with the two C are fixed on the same plane known as the peptide plane The whole plane may rotate around the N C bond phi l C Cbd il angle or C C bond psi angle A peptide plane Native functional peptides 谷胱甘肽 glutathione GSH 谷胱甘肽 glutathione GSH Tripeptidethatcontainsan unusual peptide linkage between theaminegroupofcysteine whichisattachedbynormal whichisattachedbynormal peptide linkage to a glycine and thecarboxylgroupofthethecarboxylgroupofthe glutamateside chain Itisan antioxidant preventing damage to importantcellularcomponents causedbyreactiveoxygen species such as free radicals andspecies such as free radicals and peroxides 促甲状腺素释放激素 thyrotropin releasing hormone TRH Also called thyroliberin or protirelin is a tripeptide hormone that stimulates the release of thyroid stimulating hormone 促 甲状腺素 and prolactin 催乳素 by the anterior pituitary 垂 体前叶 TRH hbd li ill fth ttt f体前叶 TRH has been used clinically for the treatment of spinocerebellar degeneration 脊髓小脑变性 and disturbance of consciousness 意识障碍 in humansdisturbance of consciousness 意识障碍 in humans The Molecular Structure of Protein How to Describe Protein Structure Primary Amino acid sequence Secondary Alpha helix beta sheet and loops Tertiary The three dimensional 3D structure Quaternary Arrangement of several polypeptide chains Quaternary Arrangement of several polypeptide chains Primary StructurePrimary Structure Thdfiid ii iill did The order of amino acids in a protein is genetically determined Contains all the information to assume its correct 3 D structure Secondary structureSecondary structure helix 螺旋 helix 螺旋 sheet 折叠 折叠 turn 转角 random coil 无规卷曲 helix helix helix is the most popular secondary helix is the most popular secondary structure elements in proteins most of the helices are right handed Every backbone N H group formes a hydrogen bond to the backbone C O group of the amino acid four residues earlierresidues earlier Each helix contains 3 6 AAs Thit h i 0 54 The pitch is 0 54nm Prediction of a Helix by it s a a squence Amino acid propensities Different amino acid sequences have different propensities for forming helical structure Methionine alanine leucine uncharged glutamate and lysine MALEK in the amino acid 1 letter codes all have especially high helix forming propensities g pp Proline and glycine have poor helix forming propensities Proline cannot donate an amide hydrogen bond having no amide hydrogen and yg gyg its sidechain interferes sterically with the backbone of the preceding turn inside a helix However proline is often seen as the first residue of a helix presumably due to its structural rigidity At the other extreme glycine also tends to disrupt helices because its high conformational flexibility makes it entropically expensive to adopt the relatively constrained helical structure sheet Amino acid propensities Large aromatic residues Tyr Phe and Trp and branched amino acids ThrLarge aromatic residues Tyr Phe and Trp and branched amino acids Thr Val Ile are favored to be found in strands in the middle of sheets sheet sheet hairpin Common sheet structural motifs Greek key motif The Greek key motif consists of four adjacent antiparallel strands and their li killinking loops meander motif A simple supersecondary protein topology composed of 2 or moreA simple supersecondary protein topology composed of 2 or more consecutive antiparallel strands linked together by hairpin loops Psi loop motifPsi loop motif The psi loop loop motif consists of two antiparallel strands with one strand in between that is connected to both by hydrogen bonds hairpin Greek key motif meander motif Psi loop motif turn turn It turns for about 180o Itt i4 AATh fi t It contains 4 AAs The first and the forth amino acid form H bond Helix Sheet composites in spider silkpp The measurement of protein electronic circular dichroism CD The measurement of protein electronic circular dichroism CD 园二色光谱 provides a probe of protein secondary stucture Motif 基序 Motif 基序 The motif is a characteristic domain structure consisting of two or more secondary structures which appears in a variety of proteins Examples helix loop helix leucine zipper zinc finger coiled coil etc Helix loop helix 螺旋 环 螺旋Helix loop helix 螺旋 环 螺旋 Two helices connected by a looploop A common motif in transcription factors e g c myc HIF MyoDfactors e g c myc HIF MyoD The smaller helices form dimers The larger helix contains theThe larger helix contains the DNA binding regions Leucine zipper 亮氨酸拉链Leucine zipper 亮氨酸拉链 Pll l l h h li Parallel alpha helices A leucine residue at every seventh position in eachseventh position in each helix One basic region for DNA binding A common motif in transcription factors c juntranscription factors c jun c fos Zinc finger 锌指 CZinc finger 锌指Cys His Two antiparallel strands and an helix His Consensus sequence Cys X2 4 Cys X3 Phe X5 Leu X2 His X3 HisX2 His X3 His Many transcription factors regulatory proteins and other proteins that interact with DNA contain zinc fingersg helix of each finger contacts the DNA Tertiary StructureTertiary Structure The different sections of h li htth helix sheet other minor secondary structure and connecting loops of a polypeptide fold in three dimensions Shape Determining Interactions in ProteinsShape Determining Interactions in Proteins 非共价结合 Noncovalent interaction 非共价结合 hydrogen bonds 氢键 N H O van der Waals forces 范德华力 hydrophobic interactions 疏水作用hydrophobic interactions 疏水作用 electrostatic salt bridges 离子键 Co alent intera tion 共价结合 Covalent interaction 共价结合 disulfide bonds 二硫键 Protein Domains 结构域 A protein domain is a part of protein sequence and structure that can evolve function and exist independently of the rest of thecan evolve function and exist independently of the rest of the protein chain 结构上及功能上 Built from different combinations of secondary structure elements and motifs Quaternary StructureQy quaternary structure is the arrangement of multiple folded iiliillil ibiprotein or coiling protein molecules in a multi subunit complex Arrangement of several polypeptide chains Each polypeptide chain is called subunit 亚基 Each polypeptide chain is called subunit 亚基 Hydrogen bonds and electrostatic salt bridges are major forces Advantages of the quaternary structureAdvantages of the quaternary structure It allows very large protein molecules to be made such as collagen 大分子形成 g It can provide greater functionality to a protein by combining different activities into a single entity 具有combining different activities into a single entity 具有 多种功能 The interactions between the subunits can often be modified by binding of small molecules and lead to the allosteric effects 变构效应 Quaternary Structure of Hemoglobin 血红蛋白 Study of Protein Structure ContentsContents Si 2 pellet after 50 NH4 2SO4 precipitation Light chain 1 antiserum 2 pellet after 50 NH4 2SO4 precipitation 3 supernanant after 50 NH4 2SO4 precipitation Isoelectric focusing 等电聚焦 Isoelectric focusing 等电聚焦 2 Dimentional Electrophoresis 2 D 双相电泳 Ion exchange chromatography 盐离子交换层析 Affinity chromatographyygp y 亲和层析 Enzyme substrate binding GST 谷胱甘肽转移酶 GST 谷胱甘肽转移酶 Biotin Avidin His 6 Ni Receptor ligand binding Antibody antigen bindingygg HA tag antibody Myc tag antibody Part II Primary structure analysisPart II Primary structure analysis Sequencing Mass spectrometry Work flow for protein sequencingWork flow for protein sequencing 1Break any disulfide bridges in the protein 破坏二硫键 1 Break any disulfide bridges in the protein 破坏二硫键 2 Separate and purify the individual chains of the protein complex if there are more than one 分离多肽链 确定氨酸组3 Determine the AA composition of each chain 确定氨基酸组成 4 Determine the terminal amino acids of each chain 确定N端 C端 5 Break each chain into fragments under 50 amino acids long 降解 5 Break each chain into fragments under 50 amino acids long 降解 6 Separate and purify the fragments 分离纯化每个肽段 7 Determine the sequence of each fragment 确定每个肽段序列 Edman 反应 反应 8 Repeat with a different pattern of cleavage 选择令一种降解方法 重复 步骤5 7 重叠确定序列9 Construct the sequence of the overall protein 重叠确定序列 From Wikipedia the free encyclopedia Example Sequencing of insulin Frederick Sanger Nobel Prize in Chemistry 1958Nobel Prize in Chemistry 1958 Mass spectrometry MS 质谱Mass spectrometry MS 质谱 Mass spectrometry is an l ilh ihanalytical technique that identifies the chemical composition of a samplecomposition of a sample based on the mass to charge ratio of chargedcharge ratio of charged particles Ion source technologies t ii t d lmatrix assisted laser desorption ionization MALDI 基质辅 助激光电离 electrospray ionization ESI 电喷射离 子化 Mlhli Mass analyzer technologies Time of flight TOF Data presentation Identify protein with MS MS Part III Tertiary structure analysisPart III Tertiary structure analysis X ray crystallography NMR NMR Bioinformatics Other methods X ray crystallography X 线晶体衍射 yygp y X ray crystallography is a method of determining the arrangement of atoms within a crystal in which aarrangement of atoms within a crystal in which a beam of X rays strikes a crystal and scatters into many different directions From the angles and ii if hd bintensities of these scattered beams a crystallographer can produce a three dimensional picture of the density of electrons within the crystalpicture of the density of electrons within the crystal From this electron density map the mean positions of the atoms in the crystal can be determined as llh ihil bdh i diddwell as their chemical bonds their disorder and sundry other information The most accurate method for protein 3D structure The most accurate method for protein 3D structure determination Crystal structure only static structure Crystal structure only static structure X ray crystallography processX ray crystallography process What happens to electron when it is hit by x rays What happens to electron when it is hit by x rays The electron starts vibrating with the same frequency as the x ray beamthe x ray beam As a result secondary beams will be scattered in all directionsdirections Primary beam Secondary beamsbeams Scattering from a molecule Molecule is composed of many electrons Each electron will scatter secondary radiation uppon exposure to x Each electron will scatter secondary radiation uppon exposure to x rays The scattered secondary beams will interact and cause interference The scattering from a molecule is dependent on number of and The scattering from a molecule is dependent on number of and distances between electrons In other words scattering from molecule is dependent on its structure If we would know the amplitudes and phases of scattered molecule we could calculate the structure of molecule Primary beam CrystallizationCrystallization Scattering from a single molecule is far too weak to be observed If molecules are all oriented in the same way the scattering from individual molecules will be multiplied in certain directions Therefore crystallization is needed Crystallization Vapor diffusion 蒸汽扩散法Vapor diffusion 蒸汽扩散法 Equilibrium dialysis 透析法 Each crystal is about 0 5 mmEach crystal is about 0 5 mm in side and contains 1012 molecules Lysozyme crystal A typical diffraction pattern from a protein crystal 1895 W C Roentgen 发现X射线 1913 W L Bragg 解析了第个小分子晶体结构 NaCl解析了第一个小分子晶体结构 NaCl 1958 J C Kendrew M F Perutz 第个蛋白结构l bi第一个蛋白结构myoglobin 1985 J Deisenhofer R Huber and 1953 J Watson and F Crick DNA双螺旋结构 H Michel 第一个膜蛋白结构 1912 M V Laue 获得x射线经过晶体后的衍射图样 DNA双螺旋结构 1840 F L H nefeld 第一个蛋白质晶体 hemoglobin 1901191419151901 Phy W C Roentgen M V Laue 1