Bài giảng Biochemistry 2/e - Chapter 5: Proteins: Their Biological Functions and Primary Structure

CHAPTER 5Proteins: Their Biological Functions and Primary Structureto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Outline5.1 Proteins - Linear Polymers of Amino Acids5.2 Architecture5.3 Many Biological Functions5.4 May be Conjugated with Other Groups5.7 Primary Structure Determination5.8 Consider the Nature of Sequences5.1 Proteins are Linear Polymers of Amino AcidsThe Peptide Bondis usually found in the trans conformationhas partial (40%) double bond characteris about 0.133 nm long - shorter than a typical single bond but longer than a double bondDue to the double bond character, the six atoms of the peptide bond group are always planar!N partially positive; O partially negativeThe Coplanar Nature of the Peptide BondSix atoms of the peptide group lie in a plane!“Peptides”Short polymers of amino acidsEach unit is called a residue2 residues - dipeptide3 residues - tripeptide12-20 residues - oligopeptidemany - polypeptide“Protein”One or more polypeptide chains One polypeptide chain - a monomeric protein More than one - multimeric protein Homomultimer - one kind of chain Heteromultimer - two or more different chains Hemoglobin, for example, is a heterotetramer It has two alpha chains and two beta chainsProteins - Large and SmallInsulin - A chain of 21 residues, B chain of 30 residues -total mol. wt. of 5,733Glutamine synthetase - 12 subunits of 468 residues each - total mol. wt. of 600,000Connectin proteins - alpha - MW 2.8 million!beta connectin - MW of 2.1 million, with a length of 1000 nm -it can stretch to 3000 nm!The Sequence of Amino Acids in a Proteinis a unique characteristic of every proteinis encoded by the nucleotide sequence of DNAis thus a form of genetic informationis read from the amino terminus to the carboxyl terminusThe sequence of ribonuclease A5.2 Architecture of ProteinsShape - globular or fibrousThe levels of protein structure - Primary - sequence - Secondary - local structures - H-bonds - Tertiary - overall 3-dimensional shape - Quaternary - subunit organizationWhat forces determine the structure?Primary structure - determined by covalent bondsSecondary, Tertiary, Quaternary structures - all determined by weak forcesWeak forces - H-bonds, ionic interactions, van der Waals interactions, hydrophobic interactionsHow to view a protein?backbone onlybackbone plus side chainsribbon structurespace-filling structureConfiguration and conformation are not the same5.3 Biological Functions of ProteinsProteins are the agents of biological functionEnzymes - RibonucleaseRegulatory proteins - InsulinTransport proteins - HemoglobinStructural proteins - CollagenContractile proteins - Actin, MyosinExotic proteins - Antifreeze proteins in fishThe tetrameric structure of hemoglobin5.4 Other Chemical Groups in ProteinsProteins may be "conjugated" with other chemical groupsIf the non-amino acid part of the protein is important to its function, it is called a prosthetic group.Be familiar with the terms: glycoprotein, lipoprotein, nucleoprotein, phosphoprotein, metalloprotein, hemoprotein, flavoprotein.5.7 Sequence DeterminationFrederick Sanger was the first - in 1953, he sequenced the two chains of insulin.Sanger's results established that all of the molecules of a given protein have the same sequence.Proteins can be sequenced in two ways: - real amino acid sequencing - sequencing the corresponding DNA in the geneInsulin consists of two polypeptide chains, A and B, held together by two disulfide bonds. The A chain has 21 residues and the B chain has 30 residues.The sequence shown is that of bovine insulin.Determining the Sequence An Eight Step Strategy1. If more than one polypeptide chain, separate.2. Cleave (reduce) disulfide bridges3. Determine composition of each chain4. Determine N- and C-terminal residuesDetermining the Sequence An Eight Step Strategy5. Cleave each chain into smaller fragments and determine the sequence of each chain6. Repeat step 5, using a different cleavage procedure to generate a different set of fragments.Determining the Sequence An Eight Step Strategy7. Reconstruct the sequence of the protein from the sequences of overlapping fragments8. Determine the positions of the disulfide crosslinksStep 1:Separation of chainsSubunit interactions depend on weak forcesSeparation is achieved with: - extreme pH - 8M urea - 6M guanidine HCl - high salt concentration (usually ammonium sulfate)Step 2:Cleavage of Disulfide bridgesPerformic acid oxidationSulfhydryl reducing agents - mercaptoethanol - dithiothreitol or dithioerythritol - to prevent recombination, follow with an alkylating agent like iodoacetateStep 3:Determine Amino Acid Compositiondescribed on pages 112,113 of G&Gresults often yield ideas for fragmentation of the polypeptide chains (Step 5, 6)Step 4:Identify N- and C-terminal residuesN-terminal analysis:Edman's reagentphenylisothiocyanatederivatives are phenylthiohydantionsor PTH derivativesStep 4:Identify N- and C-terminal residuesC-terminal analysis Enzymatic analysis (carboxypeptidase)Carboxypeptidase A cleaves any residue except Pro, Arg, and LysCarboxypeptidase B (hog pancreas) only works on Arg and LysSteps 5 and 6:Fragmentation of the chainsEnzymatic fragmentationtrypsin, chymotrypsin, clostripain, staphylococcal proteaseChemical fragmentationcyanogen bromideEnzymatic FragmentationTrypsin - cleavage on the C-side of Lys, ArgChymotrypsin - C-side of Phe, Tyr, TrpClostripain - like trypsin, but attacks Arg more than LysStaphylococcal protease C-side of Glu, Asp in phosphate bufferspecific for Glu in acetate or bicarbonate bufferChemical FragmentationCyanogen bromideCNBr acts only on methionine residuesCNBr is useful because proteins usually have only a few Met residuessee Fig. 5.21 for mechanismbe able to recognize the results!a peptide with a C-terminal homoserine lactoneStep 7:Reconstructing the SequenceUse two or more fragmentation agents in separate fragmentation experimentsSequence all the peptides produced (usually by Edman degradation)Compare and align overlapping peptide sequences to learn the sequence of the original polypeptide chainReconstructing the SequenceCompare cleavage by trypsin and staphylococcal protease on a typical peptide:Trypsin cleavage: A-E-F-S-G-I-T-P-K L-V-G-K Staphylococcal protease: F-S-G-I-T-P-K L-V-G-K-A-EReconstructing the SequenceThe correct overlap of fragments: L-V-G-K A-E-F-S-G-I-T-P-K L-V-G-K-A-E F-S-G-I-T-P-KCorrect sequence: L-V-G-K-A-E-F-S-G-I-T-P-KSequence analysis of catrocollastatin-C, a 23.6 kD protein from the venom of Crotalus atroxNature of Protein SequencesSequences and composition reflect the function of the proteinMembrane proteins have more hydrophobic residues, whereas fibrous proteins may have atypical sequencesHomologous proteins from different organisms have homologous sequencese.g., cytochrome c is highly conservedPhylogeny of Cytochrome cThe number of amino acid differences between two cytochrome c sequences is proportional to the phylogenetic difference between the species from which they are derivedThis observation can be used to build phylogenetic trees of proteinsThis is the basis for studies of molecular evolutionLaboratory Synthesis of PeptidesStrategies are complex because of the need to control side chain reactionsBlocking groups must be added and later removeddu Vigneaud’s synthesis of oxytocin in 1953 was a milestoneBruce Merrifield’s solid phase method was even more significantSolid Phase SynthesisCarboxy terminus of a nascent peptide is covalently anchored to an insoluble resinAfter each addition of a residue, the resin particles are collected by filtrationAutomation and computer control now permit synthesis of peptides of 30 residues or more