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Protein Structure(蛋白质结构与功能)Lu Linrong （鲁林荣）PhDLaboratory of Immune Regulation, Institute of Immunology Zhejiang University，School of MedicineMedical Research Building B815-819Email: Lu.Linrong@Lu Linrong@gmail comWebsite: /llrOutline•Amino acids : properties•Protein structure:primary, secondary, tertiary, and quaternary structures methods for protein separation and purificationstudymethods to protein structuresAmino Acids and Peptides(氨基酸)gAmino Acids ): Building blocks•There are over 300 naturally yamino acids on earthOnly•20amino acidsparticipate in proteinsformation•Different side chains, R,determin the propertiesof20 amino acids.20 Amino 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: BasicClassifications of amino acids1.Nonpolar(hydrophobic) 非极性2.Polar (hydrophilic) 极性中性l(h d h l)3.Acidic 酸性4.Basic 碱性1.Nonpolar hydrophobic AAs2. Polar uncharged AAs 2P l h d AA3. Acidic AAs 3Acidic AAs4. Basic AAsSpecial AAsGlycine ：side chain is –H,veryflexible Proline ：has two covalent bondswith backbone C H Cysteine ：can form disfulfide bridge2H C O O -N H 2+C H 2C H 2AminoProperties of Acids•Amino acids have both acid and base properties 两性解离)A i id h b h id d b i(•Aromatic amino acids absorb light in the near‐ultraviolet(近紫外吸收)•Coloured by ninhydrin (茚三酮反应)•Sanger’s reaction•Edman reactionAmino acids have both acid and base properties (两性解离)pH<pI pH>pIpH=pIpI, isoelectric point, 等电点:The isoelectric point is the pH at which a particular molecule Th i l t i i t i th H t hi h ti l l l or surface carries no net electrical chargeAromatic amino acids absorb light in the near ‐ultraviolet (近紫外吸收)•Aromatic amino acids(芳香族氨基酸): Trp, Tyr,Phe y•Near ‐ultraviolet•Can be used for proteinquantitationreaction Ninhydrin (茚三酮反应)440 nm for proline , hydroxyproline 540nm for other AAs p ,y yp(羟基脯氨酸)540 nm for other AAs黄色紫色离子交换层析柱茚三酮Sanger's reaction (FDNB, DNFB) Determining the N-termial amino acid in a polypeptide chain（N-端测定）硝基氟苯（二硝基氟苯）DNPDNP-蛋白DNP-氨基酸Edman reaction (PITC,苯异硫氰酸酯)1.Peptide is coupled tophenylisothiocyanate (PITC). Thefree N-terminal amino group reactswith the carbon of thei h h b f hisothiocyanate group to give thephenylthiocarbamyl (PTC)derivative of the peptide.d i ti f th tid2.Cleavage of the PTC derivativeusing anhydrous trifluoroaceticacid(无水三氯乙酸) to give the)to give theanilinothiozolinone (ATZ)derivative of the N-terminal aminoacid, and the peptide with oneacid and the peptide with onefewer amino acid,3.The ATZ residue is converted tothe phenylthiohydantoin (PTH)the phenylthiohydantoin(PTH)amino acid. (First, the ATZderivative is hydrolyzed underq,gaqueous, acidic conditions to givethe PTC amino acid. The acid thencyclizes to give the stable PTHProtein is attached to a solid support such as a chemically modified glass disk or a porous polyvinylidene fluoride membrane in the reaction cartridge.The machine performs the coupling,cleavage,conversion and coupling cleavageidentification steps(derivatives injected into an HPLC column where its retention time is compared with that of known PTH amino acid standards.)for each amino acid residue.The protein may then be identified by searching a database to match the sequence and properties of the protein.(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 2N C CR 1OH+N C H HCOOHH 2O H 2N CC R 1C H COOH H H H 22The Peptide Plane 肽平面The atoms of the group, O=C‐N‐H, together α,H with the two Care fixed on the same plane,known as the peptide plane.The whole plane may rotatearound the N‐Cαbond (phiangle) or C‐Cαbond (psi angle).l)b d(i l)A peptide planeNative functional peptides•谷胱甘肽(glutathione, GSH)：Tripeptide that contains anunusual peptide linkage between the amine group of cysteine (which is attached by normalpeptide linkage to a glycine)and the carboxyl group of theglutamate side-chain.It is an antioxidant,preventing damage to important cellular components caused by reactive oxygen species 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 has been used clinically for the treatment of )h b d li i ll f th t t t f spinocerebellar degeneration(脊髓小脑变性) anddisturbance of consciousness(意识障碍) in humans.humansThe Molecular Structure of ProteinHow to Describe Protein Structure：Primary: Amino acid sequenceSecondary: Alpha helix, beta sheet and loopsTertiary: The three‐dimensional (3D) structureArrangement of several polypeptideQuaternary: chainsStructurePrimaryTh d f i id i i i i ll d i d The order of amino acids in a protein is genetically determinedContains all the information to assume its correct 3‐D structurestructure Secondaryα‐helix (α‐螺旋)β(β折叠)‐sheet ‐β‐turn (β‐转角)random coil (无规卷曲)α‐helix‐αhelix is the most popular secondarystructure elements in proteinsmost of the α‐helices are right‐handedEvery backbone N‐H group formes a hydrogen bond  to the backbone C=O group of the amino acid four  residues earlier „Each helix contains 3.6 AAs „The Th  pitch it h is i  0.54nm 0 54Prediction of a-Helix by it’s a.a. squenceAmino-acid propensities: Different amino-acid sequences have different propensities for forming α-helical structure. Methionine, alanine, leucine, uncharged glutamate, and lysine ("MALEK" in the amino-acid 1-letter codes) all have especially high helixforming gp propensities. p Proline and glycine have poor helix-forming propensities: Proline cannot donate an amide hydrogen y g bond ( (having g no amide hydrogen), y g ) and 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 α-helical structure.β‐sheetAmino acid propensities Large aromatic residues (Tyr (Tyr, Phe and Trp) and β-branched branched amino acids (Thr (Thr, Val, Ile) are favored to be found in β strands in the middle of β sheets.β‐sheetCommon β-sheet structural motifsβ hairpin： Greek key motif: The Greek key motif consists of four adjacent antiparallel strands and their li ki l linking loops. β-meander motif: A simple supersecondary protein topology composed of 2 or more consecutive antiparallel β-strands linked together by hairpin loops. Psi-loop Psi loop motif The psi-loop, Ψ-loop, motif consists of two antiparallel strands with one strand in between that is connected to both by hydrogen bondsβ hairpinGreek key motifβ-meander motifPsi-loop motifβ‐turn„ „It turns for about 180o It contains t i  4 AAs. AA  The Th  first fi t  and the forth amino acid  form H‐bond Helix-Sheet composites p in spider p silkThe measurement of protein electronic circular dichroism (CD, 园二色光谱) provides a probe of protein secondary stucture.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, etcHelix‐loop‐helix 螺旋‐环‐螺旋•Two α‐helices connected by aloop•A common motif in transcriptionfactors: e.g. c myc, HIF, MyoD‐•The smaller helices form dimers•The larger helix contains theDNA‐binding regionsLeucine zipper 亮氨酸拉链•Parallel alpha helicesP ll l l h h li•A leucine residue at everyseventh position in eachhelix•One basic region for DNAbinding•A common motif intranscription ‐jun,factors:c junc‐fosZinc 锌指Cysfinger CHis •Two antiparallelβ‐strandsand an α‐helix•Consensus sequence:•Cys‐X2‐4‐Cys‐X3‐Phe‐X5‐Leu‐X2‐His‐X3‐Hisregulatory proteins, andother proteins that interactwith DNA contain zincfingersg•α‐helix of each fingercontacts theDNATertiary StructureThe different sections ofα‐helix, β‐sheet, otherh li h t thminor secondarystructure and connectingloops of a polypeptidefold in three dimensionsShape‐DeterminingInteractions in Proteins•Noncovalent interaction (非共价结合)hydrogen bonds 氢键N‐H…Ovan der Waals forces 范德华力hydrophobic interactions 疏水作用electrostatic salt bridges 离子键•Covalent interaction 共价结合)Co alent intera tion(disulfide bonds 二硫键Protein Domains (结构域)A protein domain is a part of protein sequence and structure thatevolve, function, independently rest of the can evolve function and exist of theprotein chain(结构上及功能上)Built from different combinations of secondary structure elements and motifsQ yQuaternary Structurequaternary structure is the arrangement of multiple folded protein or coiling protein molecules in a multi‐subuniti ili i l l i l i b i complex•Arrangement of several polypeptide chains •Each polypeptide chain is called subunit (亚基)•Hydrogen bonds and electrostatic salt bridges are major forcesAdvantages of thequaternary structureIt allows very large protein molecules to be made, such g)as collagen (大分子形成It can provide greater functionality to a protein by 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)。