Sequence determinations

The order or sequence of individual amino acid residues along the peptide chain defines the covalent structure of the molecule. It is also called primary structure in order to make a clear distinction from the three dimensional geometry in peptides and proteins. The latter is generated by non-covalent forces such as hydrogen bonds between amide groups secondary structure) and combination of polar and non-polar interactions and of disulfide bridges which result in chain folding tertiary structure). 

After cleaving the polypeptide chains into sufficiently small fragments, these must be separated by chromatography or electrophoresis and sequenced individually. The method of choice is Edman degradation, as described in section 7.2.1. 

An example of a reaction on a solid-phase matrix, the Merrifield resin, is shown in Fig. 7.14. The peptide fragments are covalently bonded, either onto a poiymer membrane or onto micrometer sized beads. The solid support is then immersed into a liquid phase and Edman degradation is carried out by sequentially adding the required reagents and removing the products for analysis. 

For gas-phase sequencing, the sample is immobilised onto a chemically inert glass frit, often by using a carrier material such as polybrene. Reagents are carried in a gaseous stream of argon and delivered to the glass frit in minute 

Hydrolysis of a peptide treated this way (eq. 17.11) would give the DNP derivative of the N-terminal amino acid other amino acids in the chain would be unlabeled. In this way, the N-terminal amino acid could be identified. 

Solution Each dipeptide will give one equivalent each of alanine and glycine on hydrolysis. Therefore, we cannot distinguish between them without applying a sequencing method. 

Treat the dipeptide with 2,4-dinitrofluorobenzene and then hydrolyze. If the dipeptide is alanylglycine, we will obtain DNP-alanine and glycine if the dipeptide is glycylalanine, we will get DNP-glycine and alanine. 

PROBLEM 17.15 Write out equations for the reactions described in Example 17.7. 

Sanger used his method with great ingenuity to deduce the complete sequence of insulin, a protein hormone with 51 amino acid units. But the method suffers in that it identifies only the N-terminal amino acid. 

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Ultimately a plasmid is defined by its mode of DNA repHcation. DNA repHcation is initiated at a single, characteristic sequence, termed the origin. The origin sequence determines the copy number of the plasmid relative to the host chromosome and the host enzymes that are involved in plasmid repHcation. Two different plasmids that contain the same origin sequence are termed incompatible. This term does not refer to the active exclusion of one plasmid by another from the ceU but rather to a stochastic process by which the two plasmids are partitioned differentially into progeny ceUs. A ceU which contains two plasmids of the same incompatibiHty group segregates two clonal populations, each of which has one of the two plasmids in it. 

The group of peptides known as tachykinins include substance P, substance K or neurokinin A, and neuromedin K, ie, neurokinin B, as well as a number of nonmammalian peptides. All members of this family contain the conserved carboxy-terrninal sequence Phe-X-Gly-Leu-Met-NH2, where X is an aromatic, ie, Phe or Tyr, or branched aliphatic, eg, Val or lie, amino acid. In general, this C-terminal sequence is cmcial for tachykinin activity (33) in fact, both the methionineamide and the C-terminal amide are cmcial for activity. The nature of the X residue in this sequence determines pharmacological identity (34,35) thus the substance P group contains an aromatic residue in this position, while the substance K group contains an aliphatic residue (33). 

Mass spectral fragmentation patterns of alkyl and phenyl hydantoins have been investigated by means of labeling techniques (28—30), and similar studies have also been carried out for thiohydantoins (31,32). In all cases, breakdown of the hydantoin ring occurs by a-ftssion at C-4 with concomitant loss of carbon monoxide and an isocyanate molecule. In the case of aryl derivatives, the ease of formation of Ar—NCO is related to the electronic properties of the aryl ring substituents (33). Mass spectrometry has been used for identification of the phenylthiohydantoin derivatives formed from amino acids during peptide sequence determination by the Edman method (34). 

J. C. Randall, Polymer Sequence Determination—Carbon 13 NMRMethods, Academic Press, New York, 1977. 

One example of a sequence determinant of redox potentials that has been identified in this manner is an Ala-to-Val mutation at residue 44, which causes a 50 mV decrease in redox potential (and vice versa) in the rubredoxins [68]. The mutation was identified because the sum of the backbone contributions to ( ) of residues 43 and 44 change by 40 mV due to an —0.5 A backbone shift away from the redox site. This example points out the importance of examining the backbone contributions. The corresponding site-specific mutants have confirmed both the redox potential shift [75] and the structural shift [75]. 

Even though these enzymes have no absolute specificity, many of them show a preference for a particular side chain before the scissile bond as seen from the amino end of the polypeptide chain. The preference of chymotrypsin to cleave after large aromatic side chains and of trypsin to cleave after Lys or Arg side chains is exploited when these enzymes are used to produce peptides suitable for amino acid sequence determination and fingerprinting. In each case, the preferred side chain is oriented so as to fit into a pocket of the enzyme called the specificity pocket. 

The reaction center is built up from four polypeptide chains, three of which are called L, M, and H because they were thought to have light, medium, and heavy molecular masses as deduced from their electrophoretic mobility on SDS-PAGE. Subsequent amino acid sequence determinations showed, however, that the H chain is in fact the smallest with 258 amino acids, followed by the L chain with 273 amino acids. The M chain is the largest polypeptide with 323 amino acids. This discrepancy between apparent relative masses and real molecular weights illustrates the uncertainty in deducing molecular masses of membrane-bound proteins from their mobility in electrophoretic gels. 

The result of sequence determination of an oligonucleotide as performed by the Sanger dideoxy chain termination method is displayed at right. 

Randall, J.C. Polymer Sequence Determination, Academic Press New York, 1977. 

Table 5.6 Codes used for amino acid residues and the corresponding mass losses observed from each in mass spectra used for sequence determination. From Chapman,...

Amino Acid Sequence Determines Primary Structure... 

Amino acid or cDNA sequencing Determination of amino acid sequence. 

The PE2 isoform has been purified and fully sequenced (Markovic and Joumval 1986). Using this sequence data Ray et al (1988) succeeded in isolating a clone fi om a tomato fruit cDNA library. The predicted amino acid sequence fi om this clone had high homology to the actual amino acid sequence determined for PE2 but was not identical. Subsequent screening of the tomato... 

Ray,J.,Knapp,J.,Grierson,D.,Bird,C and Schuch,W. (1988) Identification and sequence determination of a cDNA clone for tomato pectinesterase. European Journal of Biochemistry. 174. 119-124. 

Frantz B, AM Chakrabarty (1987) Organization and nucleotide sequence determination of a gene cluster involved in 3-chlorocatechol degradation. Proc Natl Acad Sci USA 84 4460-4464. 

Furthermore, the distribution of this peptide is compatible with PCP receptor densities, with highest concentrations of both ligand and receptor in areas that could be relevant to the psychotomimetic properties of PCP. Amino acid analysis of an aliquot of the purified fraction shows the peptide to contain at least 26 residues. Because the peptide is blocked at the N-terminus, enzyme fragments have been generated and purified over HPLC for sequence determination. 

Pentoney, Jr., S. L., Konrad, K. D., and Kaye, W., A single-fluor approach to DNA sequence determination using high performance capillary electrophoresis, Electrophoresis, 13, 467, 1992. 

Kraft K, Olbrich H, Majoul I, Mack M, Proudfoot A, Oppermann M. Characterization of sequence determinants within the carboxyl-terminal domain of chemokine... 

Proteomics algorithms have been developed to search databases of protein sequences for matches to short sequences determined experimentally from proteins or peptides in the laboratory,5 59 and for theoretical matches to... 

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