Protein sequencing amino acid composition

HEach protein is uniquely characterized by its amino acid composition and sequence. A protein s amino acid composition is defined simply as the number of each type of amino acid composing the polypeptide chain. To discover a protein s amino acid composition it is necessary to (1) break down the polypeptide chain into its constituent amino acids, (2) separate the resulting free amino acids according to type, and (3) measure the quantities of each amino acid. 

This is the concept of the Multildent tool [50, http //www.expasy.org/tools/ multiident/)]. Multildent first generates a list of best-matching proteins by amino acid composition, whose sequences are then digested theoretically and the resulting peptide masses matched against the experimentally obtained peptide mass fingerprint of the unknown protein. Similarly to... 

With a knowledge of the methodology in hand, let s review the results of amino acid composition and sequence studies on proteins. Table 5.8 lists the relative frequencies of the amino acids in various proteins. It is very unusual for a globular protein to have an amino acid composition that deviates substantially from these values. Apparently, these abundances reflect a distribution of amino acid polarities that is optimal for protein stability in an aqueous milieu. Membrane proteins have relatively more hydrophobic and fewer ionic amino acids, a condition consistent with their location. Fibrous proteins may show compositions that are atypical with respect to these norms, indicating an underlying relationship between the composition and the structure of these proteins. 

Implicit in the presumption of folding pathways is the existence of intermediate, partially folded conformational states. The notion of intermediate states on the pathway to a tertiary structure raises the possibility that segments of a protein might independently adopt local and well-defined secondary structures (a-helices and /3-sheets). The tendency of a peptide segment to prefer a particular secondary structure depends in turn on its amino acid composition and sequence. 

The amino acid compositions and sequences of the /3-strands in porin proteins are novel. Polar and nonpolar residues alternate along the /3-strands, with polar residues facing the central pore or cavity of the barrel and nonpolar residues facing out from the barrel where they can interact with the hydrophobic lipid milieu of the membrane. The smallest diameter of the porin channel is about 5 A. Thus, a maltodextrin polymer (composed of two or more glucose units) must pass through the porin in an extended conformation (like a spaghetti strand). 

The amino acid composition of resilin was elucidated shortly after its first description and was shown to be unique among other structural proteins found in Nature a summary of the amino acid sequence is given in Table 1. 

Montecucchi PC, de Castiglione R, Piani S, Gozzini L, Erspamer V. Amino acid composition and sequence of dermorphin, a novel opiate-like peptide from the skin of Phyllomedusa sauvagei. Int J Peptide Protein Res 1981 17 275-283. 

The data presented in Table 3, which includes the amino acid composition of baker s yeast and Candida krusei cytochrome c for comparison, show that Ustilago and Neurospora cytochrome c contain the same number of total residues. In seven instances, the number of residues of a particular amino acid/mole are identical. Thus, even in the absence of a sequence for the Ustilago cytochrome it can be concluded that this protein, unlike the siderochromes, has suffered little alteration in the progression from the Ascomycetes to the Basidiomycetes. This can be ascribed to the varying function of the two types of molecules. Cytochrome c must fit into a relatively specific slot bounded by a reductase and an oxidase and it has hence evolved much more slowly than the more freely acting transport agents where the specificity constraints are less demanding. 

Once sufficient numbers of intrinsically disordered and ordered protein sequences were collected, it became possible to compare them directly. The sequences in these databases were examined for dilferences in amino acid composition, sequence attributes, and evolutionary characteristics. 

Different protein folding classes can be identified by differences in their amino acid compositions (Nakashima et al., 1986) thus, we reasoned that, if disorder were encoded by the sequence, then regions of disorder would be analogous to a new folding class and hence should... 

The CD spectra of nine proteins in 6 M Gdm-HCl were studied by Cortijo etal. (1973). Those proteins with disulfide bridges were reduced and carboxymethylated. The spectra of individual proteins were not reported, but the range of values at wavelengths from 240 to 210 nm was given. The [0]222 values ranged from —800 to —2400 deg cm2/dmol. From this substantial variation, Cortijo etal. (1973) concluded that the proteins studied are not true random coils in 6 M Gdm-HCl, because random coils should have CD spectra essentially independent of amino acid composition and sequence. The observed variation was attributed to differences in the conformational distribution between allowed regions of the Ramachandran map or to residual interactions between different parts of the chain that are resistant to Gdm-HCl denaturation. 

On the other hand, the prediction by content approach is applicable regardless of the variety of sorting pathways. It may be applied to partial sequences, which are now massively produced day by day. In addition, this approach allows a simple and unified treatment, which is convenient for objective testing (e.g., cross validation). However, there is no guarantee that the amino acid composition of proteins in each localization site is well conserved. Even when a clear tendency is observed for a known set of proteins, it can be an artifact resulting from the deviation of data because the size of known proteins for each site is often insufficient to perform reliable statistical analyses. It is also evident that this approach cannot handle the differences among isoforms with different localization (see Section III,K,3). 

With methods for the quantitative analysis of amino acids to hand, the way was now open for the determination of amino acid sequences. Purified bovine insulin was relatively freely available. On the basis of ultracentrifugal analysis (Gutfreund and Ogston), a molecular weight of 12,000 was assumed—as it later emerged, a factor of 2 too high. One of the advantages from the choice of insulin as the protein to sequence was that tryptophan is absent. A 100% recovery of the amino acids could therefore be obtained easily by simple hydrolysis with HC1. In 1948 Tristram reported the complete amino acid composition of the protein. 

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