Amino acid sequences determined

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. 

Amino Acid Sequence Determines Primary Structure... 

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... 

In summary, the de novo isolation of the cDNAs encoding enzymes of alkaloid biosynthesis is still achieved by using a variety of classical techniques, such as protein purification followed by partial amino acid sequence determination, and by newer techniques such as proteomics coupled to functional heterologous expression. The current status of cloned cDNAs specifically related to isoquinoline alkaloid biosynthesis is schematically presented in Figure 10.8. New additions to this list will certainly be made in the future as a result of a combination of approaches both new and old. 

Disulfide bridges are, of course, true covalent bonds (between the sulfurs of two cysteine side chains) and are thus considered part of the primary structure of a protein by most definitions. Experimentally they also belong there, since they can be determined as part of, or an extension of, an amino acid sequence determination. However, proteins normally can fold up correctly without or before disulfide formation, and those SS links appear to influence the structure more in the manner of secondary-structural elements, by providing local specificity and stabilization. Therefore, it seems appropriate to consider them here along with the other basic elements making up three-dimensional protein structure. 

Many opioid receptors have now been cloned and their amino acid sequences determined (Evans et al. 1992 Kiefferetal. 1992). 

The successful expression of recombinant plant peroxidases such as HRP C has been a major focus of research in a number of laboratories. Three synthetic HRP C genes based on the amino acid sequence determined by Welinder (36, 47) were synthesized independently in order to initiate this work (63-65). A number of different expression systems have been evaluated (64, 66-73), a summary of which is presented in Table I. Refolding of recombinant HRP C isolated from inclusion... 

Different authors used RP-HPLC and UV detection to monitor peptide formation during cheese ripening [174-178], providing valuable information about proteolysis. When large hydrophobic peptide need to be separated an lEC represents the best choice [179]. Nevertheless, the identification of these peptides is essential for the complete understanding of the proteolytic process. The peptides eluted from the LC column can be subjected to ESl-MS for molecular weight determination and MS/MS for amino acid sequence determination, which allow rapid peptide identification [172]. HPLC-ESl-MS and MS/MS techniques have been successfully used for peptide mass fingerprint purposes for sequence analysis of purified albumin from Theobroma cacao seeds [180,181]. 

DNA and amino acid sequence determination and analysis, expression in Escherichia coli JM 83 [47]) [47]... 

Exactly how the amino acid sequence determines three-dimensional structure is not understood in detail, nor can we always predict function from sequence. However, protein families that have some shared structural or functional features can be readily identified on the basis of amino acid sequence similarities. Individual proteins are assigned to families based on the degree of similarity in amino acid sequence. Members of a family are usually identical across 25% or more of their sequences, and proteins in these families generally share at least some structural and functional characteristics. Some families are defined, however, by identities involving only a few amino acid residues that are critical... 

Ilg EC, Troxler H, Burgisser DM, Kuster T, Markert M, Guignard F, Hunziker P, Birchler N, Heizmann CW. 1996. Amino acid sequence determination of human S100A12 (P6, calgranulin C, CGRP, CAAF1) by tandem mass spectrometry. Biochem Biophys Res Commun 225(1) 146-150. 

Kato, I., Schrode, J., Kohr, W.J., Lakowski, M. Jr. 1987. Chicken ovomucoid Determination of its amino acid sequence, determination of the trypsin reactive site, and preparation of all three of its domains. Biochemistry 26 193-201. 

A protein molecule s amino acid sequence determines its properties. This has been shown for many proteins, which can be denatured and then refolded to re-form active protein tertiary structure. The biological translator thus has a somewhat easier task than the translation of human languages, because the mRNA and protein sequences are colinear. Important parts of the information don t rearrange from one language to another in contrast to the way, for example, that verbs occur at different positions in German and English sentences. 

Chemists have long appreciated that a protein s primary amino acid sequence determines its three-dimensional structure. It has also been known for some time that proteins are able to carry out their diversified functions only when they have folded up into compact three-dimensional structures. The protein-folding problem first gained prominence in the 1950s and 1960s, when Christian Anfinsen demonstrated that ribonuclease could be denatured (unfolded) and renatured reversibly. 

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