2 Структура и функция белка (1160071), страница 13
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Oxidationof cystine with performic acid produces two cysteicacid residues. Reduction by dithiothreitol to formcysteine residues must be followed by further modification of the reactive —SH groups to prevent reformation of the disulfide bond. Acetylation by iodoacetate serves this purpose.Disulfide bond(cystine)U.NHIHC-CH2C=OrO=CICH2-CHHNO=CHC-CH 2 -SH HS-CH 2 -CHC=OHNCysteic acidresiduesacetylationbyiodoacetateO=CNHHC—CH2—S—CH2—COCTOOC-CH2-S-CH2-CHHNC=OTable 6-7 The specificity of some importantmethods forfragmentingpolypeptide chainsTreatment*AcetylatedcysteineresiduesCleavage points1TrypsinLys, Arg (C)Submaxillarus proteaseArg(C)ChymotrypsinPhe, Trp, Tyr (C)Staphylococcus aureusV8 proteaseAsp, Glu (C)Asp-iV-proteaseAsp, Glu (N)PepsinPhe, Trp, Tyr (N)Cyanogen bromideMet (C)* All of the enzymes or reagents listed are available from commercial sources.fResidues furnishing the primary recognition point for theprotease; peptide bond cleavage occurs either on the carbonyl(C) or amino (N) side of the indicated group of amino acids.Breaking Disulfide Bonds Disulfide bonds interfere with the sequencing procedure.
A cystine residue (p. 116) that has one of its peptide bonds cleaved by the Edman procedure will remain attached to thepolypeptide. Disulfide bonds also interfere with the enzymatic orchemical cleavage of the polypeptide (described below). Two approaches to irreversible breakage of disulfide bonds are outlined inFigure 6-12.Cleaving the Polypeptide Chain Several methods can be used forfragmenting the polypeptide chain. These involve a set of enzymes(proteases) and chemical reagents that cleave peptide chains adjacentto specific amino acid residues (Table 6-7). The digestive enzyme trypsin, for example, catalyzes the hydrolysis of only those peptide bonds inChapter 6 An Introduction to Proteins„gProcedureamino acid analysisPolypeptidereact with FDNB; hydrolyze;v separate amino acidsreducedisulfidebonds151ResultACDEFGH5230132IKLMNPQ3222231ConclusionRSTVWYPolypeptide has 38amino acids.
Trypsin willcleave at one R(Arg) and two K (Lys)to give four fragments.Cyanogen bromide willcleave at two M (Met)to give three fragments.1211202,4-DinitrophenylasparaginedetectedN (Asn) is aminoterminal residue.(T-l) GASMALIKvT-2)placedat amino\—/terminus because itbegins with N (Asn).(T-3)placed at carboxylterminus because itdoes not end withR (Arg) or K (Lys).SHHScleave with trypsin;separate fragments; sequenceon sequenatorNGAAWHDFNPIDPRQCVHSDWLIACGPMTKcleave with cyanogenbromide; separate fragments;sequence on sequenator(C-i) NGAAWHDFNPIDPRGASMand (T-4), allowing(C%2) TKQCVHSDthem to be ordered.ALIKWLIACGPMVsequenceestablishedT-4(T-l)Aminoterminuseoverlaps withNGAAWHDFNPIDPRGASMALIKWLIACGPMTKQCVHSD©Figure 6—13 Fragmenting proteins prior to sequencing, and placing peptide fragments in theirproper order with overlaps.
The one-letter abbreviations for amino acids are given in Table 5—1. Inthis example, there are only two Cys residues, thuswhich the carbonyl group is contributed by either a Lys or an Argresidue, regardless of the length or amino acid sequence of the chain.The number of smaller peptides produced by trypsin cleavage can thusbe predicted from the total number of Lys or Arg residues in the original polypeptide (Fig.
6-13). A polypeptide with five Lys and/or Argresidues will usually yield six smaller peptides on cleavage with trypsin. Moreover, all except one of these will have a carboxyl-terminal Lysor Arg. The fragments produced by trypsin action are separated bychromatographic or electrophoretic methods.Carboxylterminus©one possibility for location of the disulfide bridge(black bracket). In polypeptides with three or moreCys residues, disulfide bridges can be located asdescribed in the text.152Part II Structure and CatalysisSequencing ofPeptides All the peptide fragments resulting from theaction of trypsin are sequenced separately by the Edman procedure.Ordering Peptide Fragments The order of these trypsin fragments inthe original polypeptide chain must now be determined.
Another sample of the intact polypeptide is cleaved into small fragments using adifferent enzyme or reagent, one that cleaves peptide bonds at pointsother than those cleaved by trypsin. For example, the reagent cyanogen bromide cleaves only those peptide bonds in which the carbonylgroup is contributed by Met (Table 6-7).
The fragments resulting fromthis new procedure are then separated and sequenced as before.The amino acid sequences of each fragment obtained by the twocleavage procedures are examined, with the objective of finding peptides from the second procedure whose sequences establish continuity,because of overlaps, between the fragments obtained by the first cleavage procedure (Fig. 6-13). Overlapping peptides obtained from the second fragmentation yield the correct order of the peptide fragments produced in the first.
Moreover, the two sets of fragments can be comparedfor possible errors in determining the amino acid sequence of eachfragment. If the amino-terminal amino acid has been identified beforethe original cleavage of the protein, this information can be used toestablish which fragment is derived from the amino terminus.If the second cleavage procedure fails to establish continuity between all peptides from the first cleavage, a third or even a fourthcleavage method must be used to obtain a set of peptides that canprovide the necessary overlap(s). A variety of proteolytic enzymes withdifferent specificities are available (Table 6-7).Locating Bisulfide Bonds After sequencing is completed, locating thedisulfide bonds requires an additional step.
A sample of the protein isagain cleaved with a reagent such as trypsin, this time without firstbreaking the disulfide bonds. When the resulting peptides are separated by electrophoresis and compared with the original set of peptidesgenerated by trypsin, two of the original peptides will be missing and anew, larger peptide will appear. The two missing peptides representthe regions of the intact polypeptide that are linked by a disulfidebond.Amino Acid Sequences Can Be Deducedfrom DNA SequencesAmino acidsequence (protein)Gln-Tyr-Pro-Thr-Ile-TrpDNA sequence (gene) CAGTATCCTACGATTTGGFigure 6-14 Correspondence of DNA and aminoacid sequences.
Each amino acid is encoded by aspecific sequence of three nucleotides (triplet) inDNA. The genetic code is described in detail inChapter 26.The approach outlined above is not the only way to obtain amino acidsequences. The development of rapid DNA sequencing methods (Chapter 12), the elucidation of the genetic code (Chapter 26), and the development of techniques for the isolation of genes (Chapter 28) make itpossible to deduce the sequence of a polypeptide by determining thesequence of nucleotides in its gene (Fig. 6-14). The two techniques arecomplementary.
When the gene is available, sequencing the DNA canbe faster and more accurate than sequencing the protein. If the genehas not been isolated, direct sequencing of peptides is necessary, andthis can provide information (e.g., the location of disulfide bonds) notavailable in a DNA sequence.
In addition, a knowledge of the aminoacid sequence can greatly facilitate the isolation of the correspondinggene (Chapter 28).Chapter 6 An InAmino Acid Sequences ProvideImportant Biochemical InformationThe sequence of amino acids in a protein can offer insights into itsthree-dimensional structure and its function, cellular location, andevolution. Most of these insights are derived by searching for similarities with other known sequences.
Thousands of sequences are knownand available in computerized data bases. The comparison of a newlyobtained sequence with this large bank of stored sequences often reveals relationships both surprising and enlightening.The relationship between amino acid sequence and three-dimensional structure, and between structure and function, is not understood in detail. However, a growing number of protein families arebeing revealed that have at least some shared structural and functional features that can be readily identified on the basis of amino acidsequence similarities alone.
For example, there are four major familiesof proteases, several families of naturally occurring protease inhibitors, a large number of closely related protein kinases, and a similarlarge number of related protein phosphatases. Individual proteins aregenerally assigned to families by the degree of similarity in amino acidsequence (identical to other members of the family across 30% or moreof the sequence), and proteins in these families generally share at leastsome structural and functional characteristics.
Some families are defined, however, by identities involving only a few amino acids that arecritical to a certain function. Many membrane-bound protein receptorsshare important structural features and have similar amino acid sequences, even though the extracellular molecules they bind are quitedifferent. Even the immunoglobulin family includes a host of extracellular and cell-surface proteins in addition to antibodies.The similarities may involve the entire protein or may be confinedto relatively small segments of it.
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