FUNDAMENTALS OF BIOCHEM
FUNDAMENTALS OF BIOCHEM BCH 401G
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Ms. Otho Gislason
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This 47 page Class Notes was uploaded by Ms. Otho Gislason on Friday October 23, 2015. The Class Notes belongs to BCH 401G at University of Kentucky taught by Staff in Fall. Since its upload, it has received 19 views. For similar materials see /class/228229/bch-401g-university-of-kentucky in Biochemistry at University of Kentucky.
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Date Created: 10/23/15
4 iimiw 7 mm Protein Structure Hierarchy tuna in i i A Supersecondary Structures Motifs Motifse ecurringgrotein structurai segments a Helixloophelix 7 two heiices connected by a turn b Coiledcoil 7 two amphipathic a heiices that interact in garaiiei through their hydrophobic edges c Helix bundlee eyerai aheiices that associate in an antigaraiiei manner to torm a bundie d MUnite two paraiiei p strands iinked to an interyening a heiix by two ioops ii iii Tertiary Structure Tertiary structure the oyeraii toiding or a groups such as metai ions hemesy etc Formed by nonecovaient interactions and 375 bo between the atoms which maybetar apart in the p sequence Supersecondary structure motif a combination sheets and ioops that treguentiy appear in muitipie organization that resuits from poiypeptide chain aiso inciuding prosthetic nds rimary ofheiice i proteins Supersecondary structures cunt e Hair a pt r p Meandere an antiparaiiei sheet composed or seguentia p strands connected by ioops or tur s pin 7 two adiacent antiparaiiei pstrands connected by urn 9 Greek keye 4 antiparaiiei strands strands i 2 in the middie 3 and 4 on the outer edges h asandwiche stacked p strands or sheets EU iiuiiiii J Selected DNA binding Motifs B Modular Domains 2 independentiy foided LEM ac units 1 proteins J mam Size 25 to 300 ammo acid residues 39 Domains are connected to k 4 g each other by ioops bound w by Weak hteraehon 93 be eenside aha Zincr nger Hehxrmmrhehx Basic iEuEinEZippEr Domem5 WSW e the evoi tionary conservation of These moms recognize DNAthrough speeme protein Structure rec mh eiectrostatic interactions i e Arg Lys and His mhchonai and evoiutionary DNA bi dmg protem o e bhd DNA asadime We ExampIes of coiIedcoi proteins Multiple distinct structuralfunctional ms of a proteln Basicieucme Zipper Hemaggiutmm Cohagenmp ehew Max122 3 trahschphomactor membraheiusmh bmamtem Nucieuiide ahmhg lt Dumam rbavvei Caiaiw Dumam w x Tim banei R eguiaiuvy Dumam we sandwich Diphtheria tom Pyruvate whase Other examples of Domains beta barrel Structural domains in proteins 393 Proteins are composed of one or usually multiple 0 as domains 01 J Neu 00 The modular nature of We make them 000000000 evolutionally flex ble and m allow the emergence of new LTOl z h39iiigl 7quotquot proteins Practically this means that regions of proteins homologous to other proteins often have similar functions This also often allows isolation of a domain by proteolysis or cloning with retention of structure andor modification of proteins without total loss of function General Principles for 3 structure 2 Compact very little solvent inside protein wellpacked side chains less conformational freedom of backbone More spherical than cylindrical or rodshaped 2 Bumpy surface 6 Hydrophobic interior and hydrophilic exterior 2 No knots in the chain 4 Not every proteins and every domains are folded intrinsically disordered regions Proteins with a similar function share the same functional domain 39 Er l El l E m 7 quotMIAquot cum Baslcleuclnezlpper transcriptionfactof n 5 W l ca 39239 The same Jnctional domain bHLHzip for DNA binding is found 39om many different unrelated proteins Folds Folding pattern of domains 393 Within each of the four main structural categories domains can be classi ed by characteristic folds 393 A fold is a combination of secondary structures that form the core of a domain 393 Some domains have simple folds others have more complex folds common names H t u ll munl m I39ul39vlllcl IN mm 39vm um l l39rr 955 if 39H Topology representation of domain folds as Mlnp ll 39t39 Very useful to compare related structures WWW and evolutionary relationships do Useful sites on protein fold and family based on domain structures CATH httpwww biochemucl acukbsmcathcath html Pfam httpAvwwsangeracukSoftwarePfaml SCOP httpscopmrcImbcamacukscopl Four categories of protein domains 1 All a domains consist almost entirely of 0c helices and loops 2 All B all domains contain only B sheets and non repetitive structures that link the B strands 3 Mixed aB contain supersecondary structures such as the ocBoc motif where regions of 0c helix and B strand alternate 4 a B domains consist of local clusters of 0c helices and B sheet in separate contiguous regions ofthe quot quot44 polypeptide chain Protein Structure Space Map oi Around 30000 proteins in human genome a 32quot g g 5 V t u 5 y 439 But only around 1 000 g rotein folds 1 39 4 il lx 3 Known common folds are v represented by points which r are spatially separated in a proportion to their structural dissimilarities 1 m 39 rl g 4 This helps us to if um I understand the evolutionary 1 or functional relationship among the proteins Comparison of the Primary and Tertiary Structures of Protein Reveals 39239 Close arn 39239 Differences refle an ce cle rlce resldues are ill hi Dlyergerltys Conyergent Eyolutlon Gerle dupllcatlorl and Transposable Genes Evolutionary Relationships ly related spe lrlO acld seque conse ct eyolutlonary cnange rrorn a common stral protelrl sequence s contaln protelrls Wltn yery slrnllar s llte ltlple Enzymes lrl a metabollc pathway Wltn tne slrnllar lrl tn 2 es lnyol edlntn ollovylrlg pathways llglycolytlc batn y 2 arnlrlo acld metabollsm 3 nucle tl rnetabol rn ryed Hennsax el Divergent Evolution of panbarrel Enzymes W0 sequerltlal reactlorls or nlstldlne andtrybtobnan blosyntnesls by related TlMebarrel enzyrnes 2mm abl crew 38 Mm W m lSlfrZEI How to arrive at a given function Dlyergel dlv lt evolutlorl A protelrls e protelrls nay unctlon Addltlorl or rnodular domalrls to a new protelrl e orten tnroug n gene dupllcatlorl and gene ruslon followed by ergence Convergent eyolunon B eanalogous or t elns e dlrrerent structure or dlrrerent ancestral gene but sarne enornologous me structure or ancestral gene but sllgntly dlrrerent r unctlon Mammallarl serlrl eyolutlon a and d Furlctlorlal dlyerslty lrl tnls ls dlrrerences lrl substrate speclrlclty e proteases t are a rotein Diversit Convergent and Divergent Evolution Convergent Evolution n Clvymauyulm E a 21 u sumutunacsmm and nymutrypstn are bum senne en dupep tdases ence tderttty and tnerrrutus ar e unretateu erveu yses peptrue band nyurutysrs ve tu run 0 Summstn Theysnare nu sequ These Wu enzymes are a ctassr exarnpte at run rgert evu Quaternary Structure Quaternary structure proterns wrtn a denned stotcmometry hgomers are more Stab ethan dtssoctated Subumts I Actwe Sttes can be formed by restdues from more than one cnarn trgand brndrng cnanges structure Quaternary Structure Refers to tne crganrzatrcn of5ubumts rn a protern wrtn rnuttrgte subumts an ohgomer Subumts may be rdentrcat ncrnc or drrrerent neterc a denned stotcmometry and arrangernent ubumts are netdtcgetner by many Weak nonccvatent rnteractrcns nydrcpncbrc etectrcstatrc as WeH as rntercnarn srs bonds Another example of Quaternary Structure of Multidomain Proteins HM mpunrc pmluuw H 71 protease nhtbttors a5 armer drug w retrovtr amprenavtr tndtnaw saqutnavtr optnaw etc Rhodopseudomonas photosynthetic reaction center Baseplate of bacteriophage T4 Sequences of DNA and protein oz Protein amino acid sequences can be deduced from the seguence of nucleotides in the corresponding gene oz A sequence of three nucleotides specifies one amino acid ACGT are DNA residues lgl lil 1 1 I l lil DNA Juan A r i A i T i A A 39 T i T intr Protein vaVs Ly a Scr jlu 7 Fm Vul Mm2 Then why bother with protein sequencing 00 There are many alternative splicing products thus DNA sequence alone can be ambiguousuninformative 00 Many proteins are translationally modified Protein Characterization oz Sequence 1 Amino acid composition analysis total digest then count how much of each amino acid 2 Amino acid sequencing Edman stepwise degradation cleave of one residue at a time then identify oz Other analytical methods 1 SDS PAGE 2 lsoelectric Focusing IEF 3 Mass spectrometry General Approach to Determining Amino Acid Sequence of a Protein Purify protein to homogeneity Determine AA composition optional Reduce SS bonds to SH groups 4 Produce multiple proteolytic fragments ofthe intact protein 5 Perform the Edman degradation method from the Nterminal residue 60 Cterminal sequencing using carboxypeptidase is difficult and not very effective cleaving and blocking disul de bonds 39239 Disullide bonds in proteins must be cleaved 1 To permit isolation ofthe PTHcysteine durin the procedure 2 To separate peptide chains 39239 Treatment with thiol compounds reduces the R SSR cystine bond to two cysteine R SH residues 39239 Subsequent treatment with an alkylating agent prevents reformation of 33 bonds Protease enzymes cleave speci c peptide bonds 39139 Trypsin cleaves on carboxyl side of basic residues Lys Arg 00 Chymotrypsin cleaves on carboxyl side of aromatic or bulky noncharged aliphatic residues eg Phe Tyr Trp 39239 Staphylococcus aureus V8 protease cleaves on carboxyl side ofnegatively charged residues Glu Asp 39239 Other endopeptidases such as pepsin subtilisin elastase and thermolysin cleaving blocking disul de bonds 2 meiumadhanal m m 12133 Cleavage sequencing an oligopeptide Figure 321 m u in n m m m m m t W m N Hm W m u m in W w m m M M w m w W Mquot um u Q m r n my Protein cleavage by a chemical reagent BrCN Figure 320 lllw w nrr 39239 cyanogen Bromide BrCN speci cally cleaves on carboxyl side ofMet Edman degradation protocol 1 Treat peptide with PITC which reacts with the Nterminus to form a PTCpeptide 2 Treat with tri uoroacetic acid TFA to s lectively cleave the Nterminal peptide bond 3 Separate Nterminal derivative 39om peptide 4 Convert derivative to PTHamino acid nd the remaining peptide becomes subject to the next cyc e 5 Identify a PTHamino acid chromatographically by HPLC a A typical example Trypsin39 a Kcocm cnymntrypsrn one means swoon Genecoou Then sublect to tne automated EDMAN sequence or ounneo tragrnents Whlle hlghly ernclent up to 50 cycles one can not seou an entlre proteln tyolcally rnore tnan 300 resroues oecause tne reactlon erncrency rs only around 98 g a Edman degradation procedure r rt r lutr w w 39 7PH9U Relulnlualkallnecundlllunsml n reactan Wllh addlllunal Pch n tne nextcvcleuYEdmandEmadallun Amlnuacld mentmcatrun M l t H Protein Sequencing Strategies 2 Starting from the Nterminal residue Edm degradation method can be used to sequence residues of a polypeptide with 50 amino acids ef ciency of 98 for each cycle thus at the end 098m 2 Typical proteins are too large to be sequenced completely by the Edman me hod Proteases enzymes cleaving peptide bonds and chemi al agents such as Cyanogen bromide BrCN are used to selectively cleave the protein into smaller fragments 2 By applying multiple proteases or reagents to the original protein overlapping segments can be identi ed and aligned then the entire sequence can be elucidated Protein Structure Hierarchy W mm Amino acid a Primary sequence Secondary iocal fouling Tertiary longrange folding Quaternary multimeric organization Supramolecular largescale assemblies b i i lt Regulation Signaling a Structure FUNCTION Transport 7 7 Movement Catalysis EH 7 V A 7 it C o Lodish Figure 3l Tertiary Structure 00 Tertiary structure the overall organization that results from folding of a polypeptide chain also including prosthetic groups such as metal ions hemes etc 0 Formed by noncovalent interactions and 88 bonds between the atoms which maybe far apart in the primary sequence Supersecondary structure motif a combination of helices sheets and loops that frequently appear in multiple proteins Helix loop helix coiled coil helix bundle 30c3 unit Hairpin 3 meander Greek key 3sandwich etc Domains discrete independently folded compact units Parallel twisted sheet 3 barrel oc3 Barrel 3 helix etc A Supersecondary Structures Motifs Motifs recurrinq protein structural segments a Helixloophelix two helices connected by a turn b Coiledcoil two amphipathic a helices that interact in parallel through their hydrophobic edges 0 Helix bundle several 0c helices that associate in an antiparallel manner to form a bundle d 3043 Unit two parallel 5 strands linked to an intervening a helix by two loops la llclurlnuiwrlxcln lb lecdmil o llclixhumll d Im m ll Supersecondary structures cont e Hairpin two adjacent antiparallel 3 strands connected by a Bturn f B Meander an antiparallel sheet composed of sequential 3 strands connected by loops or turns 9 Greek key 4 antiparallel strands strands 12 in the middle 3 and 4 on the outer edges h 3 Sandwich stacked 3 strands or sheets a Hmrwn n ummm 9 Greek m n Brwmhuch UH Wm WM 03 Selected DNA binding Motifs Zinc nger Helixturnhelix Basic leucine zipper 0 These motifs recognize DNA through specific electrostatic interactions ie Arg Lys and His 00 DNA binding proteins often bind DNA as a dimer Examples of coiledcoil proteins Basic leucine zipper Hemagglu iinin Collagen triplehelix transcription factor membrane fUS39On fibrous protein protein B Modular Domains 439 Independently folded compact units in proteins small I I SH3 domain kinasle 439 Domain size 25 to 300 xr domain amino acid residues 439 Domains are connected to each other by loops bound by weak interactions between side chains 439 Domains illustrate the evolutionary conservation of protein structure recurring Figure 3 12 part1 ol 2 Molecular Biology ollhe Cell 4th Edition functional and evolutionary module C A SH2 domain lla39rgelinage dqmainj Multiple distinct structuralfunctional domains of a protein Nucleotide Binding Catalytic Domain Bbarrel Domain n w w Catalytic A l Domain xx 1 1 I fa I DcBk I ra Receptor Tim barrel Binding 7 Domain J Regulatory Domain Trans aBoc membrane d h Domain san WIC Diphtheria toxin Pyruvate kinase Other examples of Domains alphabeta TIM barrel beta barrel General Principles for 3 structure 0 Compact very little solvent inside protein wellpacked side chains less conformational freedom of backbone 00 More spherical than cylindrical or rod shaped 0 Bumpy surface 0 Hydrophobic interior and hydrophilic exterior 00 No knots in the chain 00 Not every proteins and every domains are folded intrinsically disordered regions Structural domains in proteins 439 Proteins are composed of one or usually multiple domains 439 The modular nature of proteins make them evolutionally flexible and allow the emergence of new proteins 0 EGF W Neu precurso TPA Practically this means that regions of proteins homologous to other proteins often have similar functions This also often allows isolation of a domain by proteolysis or cloning with retention of structure andor modification of proteins without total loss of function Proteins with a similar function share the same functional domain H P p p Expression quotTAD m bHLH Myc Egr l leraimg 435v 1P LP m n 39 50a I o acudo Max ubiquitous use resting d1 ereniiaiin iD bHLH Mad dmsrammie W ceiis lSiD bHLH I Mnl ubiquitous rm l W bHLH V MIX ubiquitous Basic leucine zipper transcription factor TAD MondoA 9W TRD iAD WBSCFH4 ubiquitous KM 00 The same functional domain bHLHzip for DNA binding is found from many different unrelated proteins Folds Folding pattern of domains 139 V thin each of the four main structural categories domains can be classified by characteristic folds 139 A fold is a combination of secondary structures that form the core of a domain 139 Some domains have simple folds others have more complex folds common names a mi mm a 15mm 95gtquot quot f 4 7 w 5E lt6 w gt34 39 a Hillcl mmml mm bl 15 mm I Q 9 Four categories of protein domains 1 All a domains consist almost entirely of 0c helices and loops 2 All 5 all domains contain only 5 sheets and non repetitive structures that link the 5 strands mme 3 Mixed all contain supersecondary structures such as the 0L50L motif where regions of 0c helix and 5 strand alternate 4 on 5 domains consist of local clusters of 0c helices and 5 sheet in separate contiguous regions of the polypeptide chain Topology representation of domain folds N3 Bond 61 a1 2001 PNAS 93 5509 339 Very useful to compare related structures and evolutionary relationships 20 Useful sites on protein fold and family based on domain structures CATH httpwwwbiochemuclacukbsmcathcathhtml Pfam httpwwwsangeracukSoftwarePfaml SCOP httpscopmrclmbcamacukscop Protein Structure Space Map 00 Around 30000 proteins in human genome 0339 But only around 1 000 protein folds 39339 Known common folds are represented by points which are spatially separated in dissimilarities a proportion to their structural i 00 This helps us to understand the evolutionary or functional relationship among the proteins Hou et al PNAS 102 36516 2005 Comparison of the Primary and Tertiary Structures of Protein Reveals Evolutionary Relationships 00 Closely related species contain proteins with very similar amino acid sequences key residues are highly conserved 00 Differences reflect evolutionary change from a common ancestral protein sequence Qt Divergent vs Convergent Evolution 00 Gene duplication and Transposable Genes 3910 Multiple Enzymes in a metabolic pathway with the similar overall structures eg TIM barrel domains are found in the enzymes involved in the following pathways 1 glycolytic pathway 2 amino acid metabolism 3 nucleotide metabolism How to arrive at a given function 4 Divergent evolution A homologous proteins proteins have same structure or ancestral gene but slightly different function E me we Parent Pareru spades speclas 1 Convergent evolution B analogous proteins different structure or different ancestral gene but same function 4 Addition of modular domains to a new protein often through gene duplication and gene fusion followed by divergence Divergent Evolution of easbarrel Enzymes 6 Two sequential reactions of histidine and tryptophan biosynthesis by related TIMbarrel enzymes A E Res dua Hm aclwny a stF A gt quot0 Lang slal 20am 0 ow a l HisA a 439 u a M 39 m 7 m m AWM we we Eskabhshmemd n a P quot 7 Wquot msn YmFachwly 55A 3 19 mp4stde 1w 5 4 mm g on Julgensela mu 1 ml TpC um jTrpF I H mm Es av erF ammy on ma we snaWnld Amman em zone HennSax et al 2001 Biol Chem 382 131520 Protein Diversity Convergent and Divergent Evolution 0 Mammalian serine proteases a b and c on right are a clear example of divergent evolution and convergent evolution a and d 1 Functional diversity in this case is differences in substrate speci city Convergent Evolution t 12 gt a Subtilisin EC 342162 b Chymotrypsin EC 34211 40 Subtilisin and chymotrypsin are both serine endopeptidases They share no sequence identity and their folds are unrelated However they have an identical threedimensionally conserved Ser HisAsp catalytic triad which catalyses peptide bond hydrolysis These two enzymes are a classic example of convergent evolution 21 Quaternary Structure 39139 Refers to the organization of subunits in a protein with multine subunits an oligomerquot 39239 Subunits may be identical homo or different hetero have a defined stoichiometry and arrangement 0 Subunits are held together by many weak noncovalent interactions hydrophobic electrostatic as well as interchain SS bonds 22 Quaternary Structure Quaternary structure proteins with a de ned stoichiometry Ginm2 hi pzhains aK 0 D Inhluimnu I i crnnmgmu gmm 1 cgtumrn39yumm m we awquot W355 i mm 39 lmulnluns Txx 1 Km L u La v Q 0 L if a x i quot339 r k s 2 5 A c u no i am 5 ProteinA F cin B Protein A Protein C Stable Complex Less stable compinx Oligomers are more stable than dissociated subunits Active sites can be formed by residues from more than one chain Ligand binding changes structure Another example of Quaternary Structure of Multidomain Proteins Active site HIV l aspartic protease 2 HIV1 protease inhibitors as antiHIV drug retrovir amprenavir indinavir saquinavir Iopinavir etc Rhodopseudomonas photosynthetic reaction center ane Baseplate of bacteriophage T4 25 Protein Characterization 390 Sequence 1 Amino acid composition analysis total digest then count how much of each amino acid 2 Amino acid sequencing Edman stepwise degradation cleave of one residue at a time then identify 0 Other analytical methods 1 SDSPAGE 2 lsoelectric Focusing IEF 3 Mass spectrometry 26 Sequences of DNA and protein 39239 Protein amino acid sequences can be deduced from the sequence of nucleotides in the corresponding gene 39239 A sequence ofthree nucleotides speci es one amino acid ACGT are DNA residues l l l l l l l l l l DNA mijAAGAGTGAACCTGTvavv Protei n Lys Serf Glu iPro i Val Then why bother with protein sequencing 39239 There are many alternative splicing products thus DNA sequence alone can be ambiguousuninformative 239 Many proteins are translationally modified General Approach to Determining Amino Acid Sequence of a Protein 1 Purify protein to homogeneity 2 Determine AA composition optional 3 Reduce S S bonds to SH groups 4 Produce multiple proteolytic fragments of the intact protein 5 Perform the Edman degradation method from the Nterminal residue 60 Cterminal sequencing using carboxypeptidase is difficult and not very effective 28 Cleaving and blocking disulfide bonds 139 Disulfide bonds in proteins must be cleaved 1 To permit isolation of the PTHcysteine during the Edman procedure 2 To separate peptide chains 00 Treatment with thiol compounds reduces the R S S R cystine bond to two cysteine RSH residues 00 Subsequent treatment with an alkylating agent prevents reformation of 88 bonds 29 Cleaving blocking disulfide bonds 3 H H 0 39 I H WNifl Iicm WNic Hicm HIC HZC 1 chHZCH0H CchlrhOH gt CHwClleH 2mercaptoethanol F r 7 2C or 39I39I39 C wwicrli w WN7CH7 W 0 0 Cysnne vesidue Cysmne residues Ab if gt H C HG 19 CHI CH c006 c009 539ndmeymelhylcysmme lemme 30 Protease enzymes cleave specific peptide bonds 39to Trypsin cleaves on carboxyl side of basic residues Lys Arg 0 Chymotrypsin cleaves on carboxyl side of aromatic or bulky noncharged aliphatic residues eg Phe Tyr Trp 00 Staphylococcus aureus V8 protease cleaves on carboxyl side of negatively charged residues Glu Asp 00 Other endopeptidases such as pepsin subtilisin elastase and thermolysin 31 Cleavage sequencing an oligopeptide Figure 321 e l a H V x39 I 3 6 mM 5 lt3 lt9 H Nimiwicooe HN7 mixril hri lyAmilH Cooe Nihpi mi i700e s 0 N747 rm 76009 HXN u emicoue 7 m fume H III Protein cleavage by a chemical reagent BrCN Figure 320 e v Hmi Gly 7mg 7 Pin 7m 7 Ly Mel Trpileli c006 Mthl e HL SCN A HQ A we HC mud honinserme lncmne Z Cyanogen Bromide BrCN speci cally cleaves on carboxyl side of Met A typical example HZNSPY KAAGH TMJ TAFGACGRlGPSW TQHG RCOOH Trypsi n H2NSPYKCOOH HzNAAGHTMTAFGACGR COOH lGPSWTQH GR COOH Chymotrypsin H2NSPYCOOH HZNKAAGHTMTAFCOOH HZNG ACGRlGPSW COOH HZN TQHGRCOOH HZNSPYKAAGHTMCOOH HZNTAFGACGRlGPSWTQHGRCOOH CNBr Then subject to the automated EDMAN sequence of purified fragments While highly efficient up to 50 cycles one can not sequence an entire protein typically more than 300 residues because the reaction efficiency is only around 98 Edman degradation protocol 1 Treat peptide with PITC which reacts with the Nterminus to form a PTC peptide 2 Treat with trifluoroacetic acid TFA to selectively cleave the Nterminal peptide bond 3 Separate Nterminal derivative from peptide 4 Convert derivative to PTHamino acid 5 Identify a PTH amino acid chromatographically by HPLC and the remaining peptide becomes subject to the next cycle 35 Edman degradation procedure lquot l l l R s Hz icnicimicnicimi 7 7n l J Phenylisothiocyanate 3 Polypeptide PITC l l 1 l l l H u Nn cn c NH CH c u 5V FTC pulypeptide anhydrnus FSCCOOH R1 0 aces l i in l H ca c NH CH c N S C Original puiypepude less its Nterminnl residue N l H Thlazolinone derivative 3 Return to alkaline conditions for n 0 reaction with additional PITC in the y af x next cycle of Edman degradation HN renresvnls a solid sumwrir Amino acid identification N C 5 PTHamino acid Protein Sequencing Strategies 0 Starting from the Nterminal residue Edman degradation method can be used to sequence residues of a polypeptide with 50 amino acids efficiency of 98 for each cycle thus at the end 09850 036 30 Typical proteins are too large to be sequenced completely by the Edman method 0 Proteases enzymes cleaving peptide bonds and chemical agents such as Cyanogen bromide BrCN are used to selectively cleave the protein into smaller fragments 0 By applying multiple proteases or reagents to the original protein overlapping segments can be identified and aligned then the entire sequence can be elucidated 37
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