2 Структура и функция белка (1160071), страница 15
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Their differences in function resultfrom differences in the composition and sequenceof their amino acids. The amino acid sequences ofpolypeptide chains can be established by fragmenting them into smaller pieces using several specificreagents, and determining the amino acid sequence of each fragment by the Edman degradation procedure. The sequencing of suitably sizedpeptide fragments has been automated. The peptide fragments are then placed in the correct orderby finding sequence overlaps between fragmentsgenerated by different methods. Protein sequencescan also be deduced from the nucleotide sequenceof the corresponding gene in the DNA.
The aminoacid sequence can be compared with the thousandsof known sequences, often revealing insights intothe structure, function, cellular location, and evolution of the protein.Homologous proteins from different speciesshow sequence homology: certain positions in thepolypeptide chains contain the same amino acids,regardless of the species. In other positions theamino acids may differ. The invariant residues areevidently essential to the function of the protein.The degree of similarity between amino acid sequences of homologous proteins from different species correlates with the evolutionary relationshipof the species.See Chapter 5 for additional useful references.A Retrospect on Proteins. Ann. N.Y.
Acad. Sci.325.A collection of very interesting articles on the history of protein research.Further ReadingProperties of ProteinsCreighton, T.E. (1984) Proteins: Structures andMolecular Properties, W.H. Freeman and Company, New York.Dickerson, R.E.
& Geis, I. (1983) Proteins: Structure, Function, and Evolution, 2nd edn, The Benjamin/Cummings Publishing Company, MenloPark, CA.A beautifully illustrated introduction to proteins.Doolittle, R.F. (1985) Proteins. Sci. Am. 253 (October), 88-99.An overview that highlights evolutionary relationships.Srinavasan, P.R., Fruton, J.S., & Edsall, J.T.(eds) (1979) The Origins of Modern Biochemistry:Structure and Function of Proteins. (1989) TrendsBiochem.
Sci. 14 (July).A special issue devoted to reviews on protein chemistry and protein structure.Working with ProteinsHirs, C.H.W. & Timasheff, S.N. (eds) (1983) Methods in Enzymology, Vol. 91, Part I: Enzyme Structure, Academic Press, Inc., New York.An excellent collection of authoritative articles ontechniques in protein chemistry. Includes information on sequencing.Chapter 6 An Introduction to ProteinsKornberg, A. (1990) Why purify enzymes? In Methods in Enzymology, Vol. 182: Guide to Protein Purification (Deutscher, M.P., ed), pp.
1-5, AcademicPress, Inc., New York.The critical role of classical biochemical methods ina new age.OTarrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. BioL Chem.250, 4007-4021.An interesting attempt to count all the proteins inthe E. coli cellPlummer, David T. (1987) An Introduction toPractical Biochemistry, 3rd edn, McGraw-Hill,London.Good descriptions of many techniques for beginning students.157The Covalent Structure of ProteinsDickerson, R.E. (1972) The structure and historyof an ancient protein. Sci. Am.
226 (April), 58-72.A nice summary of information gleaned from interspecies comparisons of cytochrome c sequences.Doolittle, R. (1981) Similar amino acid sequences:chance or common ancestry? Science 214,149-159.A good discussion of what can be learned by comparing amino acid sequences.Hunkapiller, M.W., Strickler, J.E., & Wilson,K.J. (1984) Contemporary methodology for proteinstructure determination. Science 226, 304-311.Scopes, R.K. (1987) Protein Purification: Principlesand Practice, 2nd edn, Springer-Verlag, New York.Reidhaar-Olson, J.F. & Sauer, R.T.
(1988) Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science241, 53-57.A systematic study of possible amino acid substitutions in a short segment of one protein.Tonegawa, S. (1985) The molecules of the immunesystem. Sci. Am. 253 (October), 122-131.Wilson, A.C. (1985) The molecular basis of evolution. Sci. Am. 253 (October), 164-173.1. How Many [3-Galactosidase Molecules Are Present in an E. coli Cell? E.
coli is a rod-shaped bacterium 2 ^m long and 1 jtim in diameter. When grownon lactose (a sugar found in milk), the bacteriumsynthesizes the enzyme /3-galactosidase (Mr450,000), which catalyzes the breakdown of lactose. The average density of the bacterial cell is 1.2g/mL, and 14% of its total mass is soluble protein,of which 1.0% is /3-galactosidase. Calculate thenumber of /3-galactosidase molecules in an E.
colicell grown on lactose.culate the minimum molecular weight of ribonuclease. The ribonuclease molecule contains ten lysine residues. Calculate the molecular weight ofribonuclease.4. The Size of Proteins What is the approximatemolecular weight of a protein containing 682amino acids in a single polypeptide chain?5. Net Electric Charge of Peptides A peptide isolated from the brain has the sequenceGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyDetermine the net charge on the molecule at pH3.
What is the net charge at pH 5.5? At pH 8? AtpH 11? Estimate the pi for this peptide. (Use pKavalues for side chains and terminal amino and carboxyl groups as given in Table 5-1.)6. The Isoelectric Point of Pepsin Pepsin of gastricjuice (pH ~ 1.5) has a pi of about 1, much lowerthan that of other proteins (see Table 6-5). Whatfunctional groups must be present in relativelylarge numbers to give pepsin such a low pi? Whatamino acids can contribute such groups?Problems2. The Number of Tryptophan Residues in BovineSerum Albumin A quantitative amino acid analysis reveals that bovine serum albumin contains0.58% by weight of tryptophan, which has a molecular weight of 204.(a) Calculate the minimum molecular weight ofbovine serum albumin (i.e., assuming there is onlyone tryptophan residue per protein molecule).(b) Gel filtration of bovine serum albumin givesa molecular weight estimate of about 70,000.
Howmany tryptophan residues are present in a molecule of serum albumin?3. The Molecular Weight of Ribonuclease Lysinemakes up 10.5% of the weight of ribonuclease. Cal-7. The Isoelectric Point of Histones Histones areproteins of eukaryotic cell nuclei. They are tightly158Part II Structure and Catalysisbound to deoxyribonucleic acid (DNA), which hasmany phosphate groups.
The pi of histones is veryhigh, about 10.8. What amino acids must be present in relatively large numbers in histones? Inwhat way do these residues contribute to thestrong binding of histones to DNA?8. Solubility of Polypeptides One method for separating polypeptides makes use of their differentialsolubilities.
The solubility of large polypeptides inwater depends upon the relative polarity of their Rgroups, particularly on the number of ionizedgroups: the more ionized groups there are, themore soluble the polypeptide. Which of each pair ofpolypeptides below is more soluble at the indicatedpH?(a) (Gly)20 or (Glu)20 at pH 7.0(b) (Lys-Ala)3 or (Phe-Met) 3 at pH 7.0(c) (Ala-Ser-Gly) 5 or (Asn-Ser-His) 5 atpH6.0(d) (Ala-Asp-Gly) 5 or (Asn-Ser-His) 5 atpH3.09. Purification of an Enzyme A biochemist discovers and purifies a new enzyme, generating the purification table below:Procedure1.
Crude extract2. Precipitation(salt)3. Precipitation(pH)4. Ion-exchangechromatography5. Affinity chromatography6. Size-exclusionchromatographyTotalprotein(mg)Activity(units)20,0005,0004,000,0003,000,0004,0001,000,000200800,0005045750,000675,000(a) From the information given in the table, calculate the specific activity of the enzyme solutionafter each purification procedure.(b) Which of the purification procedures usedfor this enzyme is most effective (i.e., gives thegreatest increase in purity)?(c) Which of the purification procedures is leasteffective?(d) Is there any indication in this table that theenzyme is now pure? What else could be done toestimate the purity of the enzyme preparation?10.
Fragmentation of a Polypeptide Chain by Proteolytic Enzymes Trypsin and chymotrypsin arespecific enzymes that catalyze the hydrolysis ofpolypeptides at specific locations (Table 6-7). Thesequence of the B chain of insulin is shown below.Note that the cystine cross-linkage between the Aand B chains has been cleaved through the actionof performic acid (see Fig. 6-12).Phe-Val-Asn-Gln-His-Leu-CysSO 3 -GlySer-His-Leu-Val-Glu-Ala-Leu-Tyr-LeuVal-CysSOs-Gly-Glu-Arg-Gly-Phe-PheTyr-Thr-Pro-Lys-AlaIndicate the points in the B chain that are cleavedby (a) trypsin and (b) chymotrypsin. Note thatthese proteases will not remove single amino acidsfrom either end of a polypeptide chain.11. Sequence Determination of the Brain PeptideLeucine Enkephalin A group of peptides that influence nerve transmission in certain parts of thebrain has been isolated from normal brain tissue.These peptides are known as opioids, because theybind to specific receptors that bind opiate drugs,such as morphine and naloxone.
Opioids thusmimic some of the properties of opiates. Some researchers consider these peptides to be the brain'sown pain killers. Using the information below, determine the amino acid sequence of the opioid leucine enkephalin. Explain how your structure isconsistent with each piece of information.(a) Complete hydrolysis by 1 M HC1 at 110 °C followed by amino acid analysis indicated the presence of Gly, Leu, Phe, and Tyr, in a 2:1:1:1 molarratio.(b) Treatment of the peptide with l-fluoro-2,4dinitrobenzene followed by complete hydrolysisand chromatography indicated the presence of the2,4-dinitrophenyl derivative of tyrosine.
No freetyrosine could be found.(c) Complete digestion of the peptide with pepsin followed by chromatography yielded a dipeptide containing Phe and Leu, plus a tripeptide containing Tyr and Gly in a 1:2 ratio.12. Structure of a Peptide Antibiotic from Bacillusbrevis Extracts from the bacterium Bacillusbreuis contain a peptide with antibiotic properties.Such peptide antibiotics form complexes withmetal ions and apparently disrupt ion transportacross the cell membrane, killing certain bacterialspecies. The structure of the peptide has been determined from the following observations.(a) Complete acid hydrolysis of the peptide followed by amino acid analysis yielded equimolaramounts of Leu, Orn, Phe, Pro, and Val.
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