生物化學(xué)：Chapter 4 Protein structure

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1、Chapter 4 Protein StructureBiochemistry lecture for Sept. 20, 2012Most Chinese know Protein (蛋白質(zhì)）as “albumin of egg white”Proteins are structurally and functionally versatileSignal transductionPhotosynthesisCatalysisImmuneresponse DNA replicationATP synthesisMuscle contractionProteins were found to

2、be discrete chemical entities (1920s) Proteins (hemoglobin, urease, etc.) were crystallized (forming ordered arrays of identical molecular units), suggesting presence of unique structures. These observations revolutionized the thinking about proteins.Crystals ofhemoglobinCrystals ofureaseProtein str

3、ucture can be understood at four levelsProtein structure dictates function!Amino acid composition of a peptide hydrolysate can be identified using ion-exchange chromatographyRelative amino acid composition gives a characteristic profile for proteins, and often sufficient for identification of a prot

4、ein. Useful information for amino acid sequencing (e.g. choosing proteases).A standard procedure for protein hydrolysis: 6 M HCl, 24 hours, 110C). Hydrolysed samples are then derivatized (e.g., react with ninhydrin) for sensitive detection, separated by HPLC. NinhydrinAmino acid (or peptide)Purple p

5、roductA thumbprint stainedwith nihhydrinInsulin found to possess precise amino acid sequences (Sanger, 1940s-50s) Partial hydrolysis (Acid, enzymes) and separation of short peptides; Identification of N-terminal residues (DNP labeled) and amino acid composition; disulphide bridge split with performi

6、c acid to separate two chains; milder acid treatment (some peptide bonds not split) No periodicity, similar among different species.Frederick Sanger(1918-) Identification of DNP-amino acidsvia partition chromatography.Deduction of sequences of cysteic acid peptidesPolypeptide can be specifically cle

7、aved into shorter peptides by proteases or chemical reagentsRoom Temp.Acidic A protease (proteinase, proteolytic enzyme)cleaves peptide bonds of certain featuresA typical procedure for amino acid sequencingProtein sequence determination methods: tandem mass spectrometry Mass of a protein can be prec

8、isely determined by Matrix-assisted laser desorption-ionization (MALDI) or electrospray spectrometry.MALDI-TOF (Time of flight)Mass analyzer Koichi Tanaka et al. (1988). Protein and Polymer Analyses up to m/z 100 000 by Laser Ionization Time-of flight Mass Spectrometry. Rapid Commun Mass Spectrom 2

9、(20): 1513. Koichi Tanaka(Nobel prize 2002) No Ph.D.Not a ProfessorProtein sequence determination methods: tandem mass spectrometry The difference in mass from peak to peak identifies the amino acid that was lost in each case, thus revealing the sequence of the peptide.The successive peaks differ by

10、 the mass of a particular amino acid in the original peptide.PheProGlyGln(Ile/Leu)AsnAlaAsp(Ile/Leu)ArgJohn B. Fenn (Nobel prize 2002)Achieved after his retirement from Yale! Fenn et al., (1989) Electrospray Ionization for Mass Spectrometry of Large Biomolecules, Science, 246: 64-71 Applications:Typ

11、es of proteins present from a sample;Posttranslational modifications;Protein-protein interactions;Biomarker identification for diseasesProteomics: large-scale characterization of proteins, combining high resolution separation of proteins (2-D gel or LC), Mass spectrometry measurement, and bioinforma

12、tics data base search.Partial double bond character of peptide bonds revealed by Pauling and Corey (1930s-40s) X-ray structure of several amino acids and dipeptides: bond length is unique. The peptide bond is planar. 0.132Partial double bond feature:Single-bonded form: 60%);Double-bonded form: 40%).

13、 Peptide bonds in proteins are in trans configuration except the X-Pro linkages The trans form is strongly favored because of steric clashes that occur in the cis form. For X-Pro: the cis and trans isomers being nearly equal in energy Peptidylprolyl isomerase (PPI) Ca aCa aCa aCa aCa aCa aCa aCa aTh

14、e rotation of the single bonds along the peptide backbone is restricted by steric clashes: the Ramachandran plot G.N. Ramachandran J. Mol. Biol., (1963) 7: 95-99 As one looks out from the a carbon, the f (phi, N-Ca) and (psi, Ca-C) angles increase as the carbonyl or amide nitrogens (respectively) ro

15、tate clockwise.+ or - 180o allowedBoth f f and = 0o a prohibited conformationIntra-chain Hydrogen bonding patterns of the backbone predicted (1951, Pauling and Corey)Pauling et al., (1951) The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. PNAS., (1951) 3

16、7: 205-211 William Astbury (1930s): unstretched protein molecules formed a helix (the -form); the stretched form an extended state (the -form); based on X-ray studies of hair & wool.Astbury(1898-1961)Pauling(1901-1994)Corey(1897-1971)XModel builtOn measured:bond lengths;bond angles;configurations.Ba

17、ckboneN-O hydrogenbonding at 2.72 A;All residuesstereochemicallyequivalent(A left-handedHelix builtwith D-amino acids)3.7 residuesper turn5.1 residuesper turnResidue iResidue i-4Inter-chain hydrogen bonding patterns of the backbone predicted (1951, Pauling and Corey)Pauling and Corey (1951) The plea

18、ted sheet, a new layer configuration of polypeptide chains. PNAS, 37:251-256. Opposite orientationSimilar orientationLayer configurationSecondary structure: highly regular local sub-structures a a-helices and b b-sheet structures are common.In addition, b b-turn structures (four residues, often cont

19、aining Gly and Pro) are also commonly found in proteins.movement away from the observer is clockwise movement is in the anti-clockwisedirection Only right-handed helices found in proteins (myoglobin, 1961)Thumb points along the helix axis, helix turns in the direction indicated by the fingers.,Amino

20、 Acids Have Different Propensities for Forming Alpha Helices, Beta Sheets, and Beta TurnsSecondary structure can be predicted based on the amino acid sequence (up to 80% accuracy).Amino acid helix sheetTurnAla1.290.900.78Cys1.110.740.80Leu1.301.020.59Met1.470.970.39Glu1.440.751.00Gln1.270.800.97His1

21、.221.080.69Lys1.230.770.96Val0.911.490.47Ile0.971.450.51Phe1.071.320.58Tyr0.721.251.05Trp0.991.140.75Thr0.821.211.03Gly0.560.921.64Ser0.820.951.33Asp1.040.721.41Asn0.900.761.28Pro0.520.641.91Arg0.960.990.88Secondary structures can be assessed by circular dichroism spectroscopy Circular dichroism (CD

22、): differential absorption of left-handed and right-handed plane-polarized light for optically active chiral (asymmetric) molecules. Particular asymmetric structures exhibit a characteristic CD spectrum reflecting its unique structure (e.g., a-helix, b-sheet of proteins, DNA double helix, etc).Plane

23、-polarized lightCD spectra of typical proteins at the far UV region (peptide bonds being the chromophore)Difference in molar extinction coefficients for the two lights.3-D “structures” of myoglobin and hemoglobin determined (1930s-50s, Perutz and Kendrew) MyoglobinHemoglobin(10,000 atoms)X-ray diffr

24、action patternsStructural models65.5 HemeHemeHemeThe workflow ofX-ray crystallography Wavelength: 0.7-1.5 Each atom makesa contribution to each reflection spot.About 25, 000 reflectionsFourier transform3-D structures can also be determined by Nuclear magnetic resonance (NMR) spectroscopy (in solutio

25、n)Uses the magnetic spin properties of atomic nuclei within a molecule to identify atoms that are close together in space (either because they are bonded together or because folds of a protein chain bring them together).The nuclear Overhauser effect (NOE) identifies pairs of atoms that are in close

26、proximity. 3-D structure of proteins is reconstructed using such distance constrains.Richard R. ErnstNobel prize in 1991 Kurt WthrichNobel Prize 2002 2-D 15N, 1H heteronuclear single quantum correlation (HSQC) NMR spectrum: Plots of correlation chemical shift peaks between all the amide nitrogen and

27、 their protons (being assigned to the individual residues of the protein) Contour plotsof correlationsbetween amide N& their HCommon steps of NMR protein structure determinationChemical shifts reflecting correlations of 1H,1H (homonuclear) and 1H,13C or 1H,15N (heteronuclear) often measured.Correlat

28、ions many indicate whether two nuclei share a through-bond or through-space characteristic.Dynamic structure can be revealed (intrinsically disordered structure also revealed).Protein-ligand interaction can be examined (useful for drug screening. An iterative processEnsemble of multiple structures o

29、ften displayed.Tertiary structure: The spatial arrangement of all atoms in a protein Both a-helices (only the right-handed one) and b-sheets observed in the 3-D structure of proteins. Soluble proteins: Driven by non-specific hydrophobic interactions (burying hydrophobic residues from H2O); Stabilize

30、d by specific tertiary interactions, e.g., salt bridges, H bonds, tight packing between the side chains, (disulfide bonds for excreted proteins)Ribonuclease APorinMyoglobinWith hydrophobic interiorWith hydrophilic interiorEach protein possess one or a few native conformations usually with marginal s

31、tability Native conformation: the tertiary structure of a protein under physiological conditions. Protein tertiary structure, maintained by non-covalent weak interactions, is subject to unfolding (denaturization) when conditions become non-optimal (as for acidic pH or high temperature). Unfolded pro

32、teins tend to form aggregates.DisorderedaggregateNative conformationAmyloidfiberProtein denaturization was considered as the unfolding of its 3-D structures (Tsien Wu, 1931) Protein denaturation was a purely conformational change, i.e., corresponded to protein unfolding and not to some chemical alte

33、ration of the protein (Tsien Wu,吳憲). Wu, H. (1931) “Studies on the denaturization of proteins, 13: A theory of denaturization. Chinese Journal of Physiology 5:321-344. Rpt. Adv. Protein Chem., 46:6-26 (1995).Mirsky and Pauling (1936) “On the Structure of Native, Denatured, and Coagulated Proteins. P

34、NAS, 22: 439447 吳憲吳憲 （1893-1959）Quaternary structure: larger assembly of several protein molecules (subunits) Stabilized by the same non-covalent interactions as found in 3-D structures. Homo- or hetero-oligomers can be formed. Proteins are nanoparticles (definition: 1100 nm).HemoglobinATP synthaseV

35、iral capsids. Reversible and substrate induced activation of DegP protease-chaperone via homo-oligomerization to function as molecular chaperone and protease: a working model.Homo-oligomerization serves as a Common Way for Proteins to Modulate their ActivtiesCertain sub-structural patterns recur in

36、different (often unrelated) proteins The structural and sequence motifs (super-secondary units, folds): Common folding patterns made of two or more secondary structure elements (e.g., zinc finger, b barrel, b-a-b loop, etc) that are repeatedly used by a variety of proteins. A structural domain: The

37、multiple self-stabilizing independent 3-D entities present in one single protein. Zinc finger CD4b-b-barrelDomain 1Domain 2Domain 3Domain 4Globular proteins can be classified according to their patterns of foldingStructural comparison of proteins allows evolution to be examined at the molecular leve

38、l Molecular phylogenies be achieved by comparing amino acid sequences of homologous and functionally important proteins (or ribosomal RNAs).EF-1a a/EF-TuMany proteins & genomeComparingGroEL (Hsp60)Proteins have different sizesThe average MW of amino acid residues: 110Longestpolypeptidechainconjugate

39、d proteins contain non-amino acid prosthetic groupsThe 3-D structure of proteins were found to be determined by their amino acid sequences (Anfinsen, 1961)Unfolded and oxidized ribonuclease A can refold intoits active form (with disulfide bonds correctly reformed)A thermodynamic hypothesis: Details

40、of the 3-D structure of a protein determined entirely by its amino acid sequence and no other outside information is required.Christian Anfinsen (1916-1995) Nobel prize, 1972There would be 105 possible disulfide bonding patterns in the scrambled proteinBUT:Protein folding in vivo often aided by mole

41、cular chaperonesProtein folding and unfolding were found to be highly cooperative process Most proteins show a sharp transition from the folded to unfolded upon treating with increasing concentrations of denaturants, and vice versa. Folding and unfolding is largely an “all or none” process results f

42、rom a cooperative transition.Protein peptide chains fold into its 3-D structure in an efficient but unknown mechanism The Levinthals Paradox (the folding problem): Very large number of degrees of freedom in an unfolded polypeptide chain leads to an astronomical number of possible conformations. sequ

43、entially sampling all the possible conformations would require a time longer than the age of the universe; Most small proteins fold spontaneously on a millisecond or even microsecond time scale. Protein folding is depicted as a free-energy funnel:Down the funnel, less free energy, more native confor

44、mation (semistable intermediates formed on The way)Prion was found to be an infectious self-reproducing agent consisting solely of proteins which converts from normal a a-helices to misfolded b b-sheets and form amyloid fibersStanley B. PrusinerHow prion proteins replicate (propagate) in a protein o

45、nly manner is still an open questionHeterodimer model a single PrPSc molecule binds to a single PrPC molecule and catalyzes its conversion into PrPScFibril dependent modelPrPSc exists only as fibrils, and that fibril ends bind PrPC and convert it into PrPScThe dynamic and flexibility aspect of prote

46、in structure is still poorly understood It is a fast and complex process and happens for newly synthesized polypeptides to fold and proteins to function. No techniques to effectively follow such processes and mostly simulated via computer modeling.Motions Within Globular Protein Molecules Lactoferri

47、nSome proteins were found to be intrinsically unfolded (unstructured) They were found to lack stable tertiary structure when exist as an isolated polypeptide chain (a subunit) under physiological conditions in vitro. Particularly enriched in proteins implicated in cell signaling, transcription and c

48、hromatin remodeling functions. Such structure is usually invisible in the crystal structure.De novo protein designs have achieved limited progresses Depends on our understanding of protein folding principle and structure-function relationship.Computational prediction of 3-D structure of proteins fro

49、m amino acid sequences remains a great challengeGenome sequencing reveals the amino acid sequences of all the putative proteins of an organism.Template-based or free modelingThe biogenesis of proteins involves translation, folding, translocation, and an effective quality control system Molecular cha

50、perones play key roles.Topics for the 3rd APPA Conference(May 6-9,2011, Shanghai, China)“Protein and Beyond”topics 1. Protein biosynthesis, trafficking and quality control 2. Protein folding, structure and dynamics 3. Protein posttranslational modification and activity modulation 4. Protein interact

51、ion, network and proteomics 5. Protein machinery and action mechanism 6. Protein design and engineering 7. Protein real time and quantitative detection, and single molecule study 8. Proteins in disease and drug discovery 9. Protein applications in biotechnology and therapeutics 10. Interdisciplinary

52、 study on proteinsAims and Scope of Protein ScienceThe Journal encompasses the structure, function, and biochemical significance of proteins, their role in molecular and cell biology, genetics, and evolution, and their regulation and mechanisms of action. Topics of particular interest include, but a

53、re not limited to:Structure of proteins and strategies of determining protein structure by chemical, biophysical, and recombinant methodsPeptidesProtein domainsProtein folding and molecular dynamicsNovel isolation proceduresEnzyme action and regulationInteractions of proteins with nucleic acids, lip

54、ids, ligands, and other proteinsReceptor-mediated signal transduction and other trans-membrane phenomenaThe functions of proteins in replication, supramolecular assembly, immune reactions, development, and other biological processesProtein trafficking, synthesis and sortingRecognition, localization

55、and signaling of proteinsQuiz 1 Pepsin is the name given to several digestive enzymes that are secreted (as larger precursor proteins) by glands that line the stomach. These glands also secrete hydrochloric acid, which dissolves the particulate matter in food, allowing pepsin to enzymatically cleave

56、 individual protein molecules. The resulting mixture of food, HCl, and digestive enzymes is known as chyme and has a pH near 1.5. What pI would you predict for the pepsin proteins and what might happen to pepsin proteins after they enter intestine where pH is relatively high？Which amino acids in the proteins would contribute functional groups to confer this pI？