2 Структура и функция белка (1160071), страница 12
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The amino acid sequence of ubiquitin is identical in species asdisparate as fruit flies and humans.Is the amino acid sequence absolutely fixed, or invariant, for aparticular protein? No; someflexibilityis possible. An estimated 20 to30% of the proteins in humans are polymorphic, having amino acidsequence variants in the human population. Many of these variationsin sequence have little or no effect on the function of the protein. Furthermore, proteins that carry out a broadly similar function in distantly related species often differ greatly in overall size and amino acidChapter 6 An Introduction to Proteinssequence. An example is DNA polymerase, the primary enzyme involved in DNA synthesis. The DNA polymerase of a bacterium is verydifferent in much of its sequence from that of a mouse cell.Table 6-6 A sampling of genetic diseases linked to loss or defect of a single enzyme or proteinDiseasePhysiological effectsAffected enzyme or proteinCystic fibrosisAbnormal secretion in lungs,pancreas, sweat glands;chronic pulmonary diseasegenerally leading to deathin children or young adultsNeurological defects, selfmutilation, mental retardationSevere loss of immuneresponseSevere loss of immune response (children must livein a sterile bubble)Erosion of bones, hip joints;sometimes brain damageOverproduction of uric acidresulting in recurring attacks of acute arthritisShort stature, convulsionsChloride channelPurine nucleosidephosphorylaseAdenosine deaminaseAtherosclerosis resulting fromelevated cholesterol levelsin blood; sometimes earlydeath from heart failureMotor weakness, mental deterioration, death by age 3 yrPain, swelling in hands andfeet; can lead to suddensevere pain in bones orjoints and deathHexosaminidase-ALesch-Nyhan syndromeImmunodeficiency diseaseImmunodeficiency diseaseGaucher's diseaseGout, primaryRickets, vitamin D-dependentFamilial hypercholesterolemiaTay-Sachs diseaseSickle-cell anemiaHypoxanthine-guaninephosphoribosyl transferaseGlucocerebrosidasePhosphoribosyl pyrophosphatesynthetase25-Hydroxycholecalciferol-lhydroxylaseLow-density lipoproteinreceptorHemoglobinThe amino acid sequence of a protein is inextricably linked to itsfunction.
Proteins often contain crucial substructures within theiramino acid sequence that are essential to their biological functions.The amino acid sequence in other regions might vary considerablywithout affecting these functions. The fraction of the sequence that iscritical varies from protein to protein, complicating the task of relatingsequence to structure, and structure to function. Before we can consider this problem further, however, we must examine how sequenceinformation is obtained.147148A chainNH3NH3GlyPheHeValValAsnIIGluGinGinHisILeuCysCysAlaGlySerSer10 Val10 HisCysLeuSerILeuValTyrAla15 GinLeuIIGluIIILeuIGluITyrLeuAsnValCysCysGlyAsnGluCOO"ArgGlyPheI25 PheITyrThrIProILys30AlaCOO"Frederick SangerFigure 6-10 The amino acid sequence of the twochains of bovine insulin, which are joined by disulfide cross-linkages. The A chain is identical inhuman, pig, dog, rabbit, and sperm whale insulins.The B chains of the cow, pig, dog, goat, and horseare identical.
Such identities between similar proteins of different species are discussed later in thischapter.II20B chainThe Amino Acid Sequence of Polypeptide ChainsCan Be DeterminedTwo major discoveries in 1953 ushered in the modern era of biochemistry. In that year James D.
Watson and Francis Crick deduced thedouble-helical structure of DNA and proposed a structural basis for theprecise replication of DNA (Chapter 12). Implicit in their proposal wasthe idea that the sequence of nucleotide units in DNA bears encodedgenetic information. In that same year, Frederick Sanger worked outthe sequence of amino acids in the polypeptide chains of the hormoneinsulin (Fig. 6-10), surprising many researchers who had long thoughtthat elucidation of the amino acid sequence of a polypeptide would be ahopelessly difficult task. These achievements together suggested thatthe nucleotide sequence of DNA and the amino acid sequence of proteins were somehow related. Within just over a decade, the nucleotidecode that determines the amino acid sequence of protein molecules hadbeen revealed (Chapter 26).Today the amino acid sequences of thousands of different proteinsfrom many species are known, determined using principles first developed by Sanger.
These methods are still in use, although with manyvariations and improvements in detail.Short Polypeptides Are SequencedUsing Automated ProceduresThree procedures are used in the determination of the sequence of apolypeptide chain (Fig. 6-11). The first is to hydrolyze it and determineits amino acid composition (Fig.
6-lla). This information is often valuable in later steps, and can also be useful in itself. Because amino acidcomposition differs from one protein to the next, it can serve as a kindof fingerprint. It can be used, for example, to help determine whetherproteins isolated by different laboratories are the same or different.Often, the next step is to identify the amino-terminal amino acidresidue (Fig. 6-llb). For this purpose Sanger developed the reagentl-fluoro-2,4-dinitrobenzene (FDNB; see Fig. 5-14). Other reagentsused to label the amino-terminal residue are dansyl chloride and dabsyl chloride (see Figs. 5-14 and 5-18). The dansyl derivative is highlyfluorescent and can be detected and measured in much lower concentrations than dinitrophenyl derivatives.
The dabsyl derivative is intensely colored and also provides greater sensitivity than thedinitrophenyl compounds. These methods destroy the polypeptide andtheir utility is therefore limited to identification of the amino-terminalresidue.6 M HC1(a)Determinetypes andamountsof aminoacids inpolypeptideHPLC or ion-exchangeFree aminochromatography^ Amino acid*acids149PolypeptideNO2N02NO 2Free+ aminoacids6 M HC1(b)2,4Dinitrophenylderivativeof polypeptideR2phenylisothiocyanate2,4-Dinitrophenylderivativeof amino-terminalresidueHNlu|C=ONH|c=sR2—CHC=O6 M HC1(c)"OHIHNR2—CHIdentifyaminoterminalresidue ofpolypeptidePhenylthiohydantoinamino acidHN3R —CHC=OIdentifyaminoterminalresidue;purifyand recycleremainingpeptidefragmentthroughEdmanprocessC=OTo sequence the entire polypeptide, a chemical method devised byPehr Edman is usually employed.
The Edman degradation procedure labels and removes only the amino-terminal residue from a peptide, leaving all other peptide bonds intact (Fig. 6-llc). The peptide isreacted with phenylisothiocyanate, and the amino-terminal residue isultimately removed as a phenylthiohydantoin derivative.
After removal and identification of the amino-terminal residue, the newamino-terminal residue so exposed can be labeled, removed, and identified by repeating the same series of reactions. This procedure is repeated until the entire sequence is determined. Refinements of eachstep permit the sequencing of up to 50 amino acid residues in a largepeptide.The many individual steps and the careful bookkeeping required inthe determination of the amino acid sequence of long polypeptidechains are usually carried out by programmed and automated analyzers. The Edman degradation is carried out on a programmed machine,called a sequenator, which mixes reagents in the proper proportions,separates the products, identifies them, and records the results. Suchinstruments have greatly reduced the time and labor required to determine the amino acid sequence of polypeptides. These methods are extremely sensitive.
Often, less than a microgram of protein is sufficientto determine its complete amino acid sequence.Figure 6—11 Steps in sequencing a polypeptide.(a) Determination of amino acid composition and(b) identification of the amino-terminal residueare the first steps for many polypeptides. Sanger'smethod for identifying the amino-terminal residueis shown here. The Edman degradation procedure(c) reveals the entire sequence of a peptide. Forshorter peptides, this method alone readily yieldsthe entire sequence, and steps (a) and (b) are oftenomitted.
The latter procedures are useful in thecase of larger polypeptides, which are often fragmented into smaller peptides for sequencing (seeFig. 6-13).150Part II Structure and CatalysisLarge Proteins Must Be Sequenced in Smaller SegmentsThe overall accuracy for determination of an amino acid sequence generally declines as the length of the polypeptide increases, especially forpolypeptides longer than 50 amino acids.
The very large polypeptidesfound in proteins must usually be broken down into pieces smallenough to be sequenced efficiently. There are several steps in this process. First, any disulfide bonds are broken, and the protein is cleavedinto a set of specific fragments by chemical or enzymatic methods.Each fragment is then purified, and sequenced by the Edman procedure. Finally, the order in which the fragments appear in the originalprotein is determined and disulfide bonds (if any) are located.Figure 6-12 Breaking disulfide bonds in proteins.The two common methods are illustrated.
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