Polypeptides amino acid sequence determination

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 cytochrome 62 core has been shown by amino acid sequence determination to be located at the N-terminus of the flavocytochrome 62 polypeptide chain (43). It is clearly seen as a distinct domain in the crystal structure, in close contact with the much larger FMN-contain-ing domain (23-25) (Fig. 5). The cytochrome domain consists of resi-... 

Proteins have a covalently bonded backbone, as discussed before, in relation to amino acid sequence determination. But the 3-D shape or conformation is held together by weaker bonding of the noncovalent type. The linear form of the polypeptide backbone of the protein folds into a tightly held shape, which is chemically stabilized by weak bonds like hydrogen bonds, ionic bonds, and hydrophobic interactions among nonpolar amino acid side chains [19]. 

The N-terminal amino acid sequence determined for the 5 kDa polypeptide is shown in Fig. 3. This sequence did not correspond to any possible reading frame in the chloroplast DNA of liverwort or tobacco, suggesting that the 5 kDa polypeptide is encoded by the nuclear DNA. Furthermore, this protein sequence did not match any chloroplast components previously reported for higher plants and cyanobacteria, indicating that the 5 kDa polypeptide is new component. Nine of the first 21 N-terminal amino acids are hydrophobic (two Phenylalanine, two isoleucine, two leucine, two methionine and one Valine). The protein contains only one charged amino acid (Aspartic acid) and is easy to extract from the gel using SDS-isopropanol. The data indicate that the 5 kDa polypeptide is a hyrophobic protein, suggesting that this protein is not located on the surface of thylakoid membranes. 

Translation The stepwise synthesis of a polypeptide with an amino acid sequence determined by the nucleotide sequence of the mRNA coding region. The genetic code relates each amino acid to a group of three... 

Much of protein engineering concerns attempts to explore the relationship between protein stmcture and function. Proteins are polymers of amino acids (qv), which have general stmcture +H3N—CHR—COO , where R, the amino acid side chain, determines the unique identity and hence the stmcture and reactivity of the amino acid (Fig. 1, Table 1). Formation of a polypeptide or protein from the constituent amino acids involves the condensation of the amino-nitrogen of one residue to the carboxylate-carbon of another residue to form an amide, also called peptide, bond and water. The linear order in which amino acids are linked in the protein is called the primary stmcture of the protein or, more commonly, the amino acid sequence. Only 20 amino acid stmctures are used commonly in the cellular biosynthesis of proteins (qv). 

Secondary structure occurs mainly as a helices and p strands. The formation of secondary structure in a local region of the polypeptide chain is to some extent determined by the primary structure. Certain amino acid sequences favor either a helices or p strands others favor formation of loop regions. Secondary structure elements usually arrange themselves in simple motifs, as described earlier. Motifs are formed by packing side chains from adjacent a helices or p strands close to each other. 

From a map at low resolution (5 A or higher) one can obtain the shape of the molecule and sometimes identify a-helical regions as rods of electron density. At medium resolution (around 3 A) it is usually possible to trace the path of the polypeptide chain and to fit a known amino acid sequence into the map. At this resolution it should be possible to distinguish the density of an alanine side chain from that of a leucine, whereas at 4 A resolution there is little side chain detail. Gross features of functionally important aspects of a structure usually can be deduced at 3 A resolution, including the identification of active-site residues. At 2 A resolution details are sufficiently well resolved in the map to decide between a leucine and an isoleucine side chain, and at 1 A resolution one sees atoms as discrete balls of density. However, the structures of only a few small proteins have been determined to such high resolution. 

X-ray structures are determined at different levels of resolution. At low resolution only the shape of the molecule is obtained, whereas at high resolution most atomic positions can be determined to a high degree of accuracy. At medium resolution the fold of the polypeptide chain is usually correctly revealed as well as the approximate positions of the side chains, including those at the active site. The quality of the final three-dimensional model of the protein depends on the resolution of the x-ray data and on the degree of refinement. In a highly refined structure, with an R value less than 0.20 at a resolution around 2.0 A, the estimated errors in atomic positions are around 0.1 A to 0.2 A, provided the amino acid sequence is known. 

Determining the Amino Acid Sequence Nature of Amino Acid Sequences Synthe.sis of Polypeptides in the Laboratory... 

Later we return to an analysis of the 1° structure of proteins and the methodology used in determining the amino acid sequence of polypeptide chains, but let s first consider the extraordinary variety and functional diversity of these most interesting macromolecules. 

Heliantholysin. The major form of heliantholysin is a basic polypeptide chain (pi in the region of 9.8) having a molecular weight of 16,600. Its amino acid sequence has been determined (11). It is powerfully hemolytic for washed erythrocytes derived from a variety of animals, those of the cat being the most sensitive, and those of the guinea pig the most resistant (10). As is true of most hemolytic systems, the biochemical basis for the very large differences in sensitivity of erythrocytes from different animal species is unknown. 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

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