Amino acid sequencing of proteins

Amino acid sequencing may be carried out in a number of ways. The most widely used is the Edman degradation procedure in which phenylisothiocyanate is used to react with the amino acid residue at the amine end of the protein chain. This derivatized residue is removed from the remainder of the protein and converted to a phenylhydantoin derivative which is identified by using, for example, HPLC. 

Amino acid a-Helix Polypeptide chain Assembled sub-units 

A series of these procedures are then carried out on the remaining protein, thus enabling each new terminal amino acid residue to be identified. 

It is not practical to sequence more than about 50 amino acid residues on a single protein in this way and larger proteins need to be broken down into polypeptides with more appropriate lengths to allow complete sequencing to be carried out. This shortening of the polypeptide chain may be carried out using chemical or enzymatic methods, cf. hydrolysis. 

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The protein folding problem is the task of understanding and predicting how the information coded in the amino acid sequence of proteins at the time of their formation translates into the 3-dimensional structure of the biologically active protein. A thorough recent survey of the problems involved from a mathematical point of view is given by Neumaier [22]. 

Recombinant DNA techniques have provided tools for the rapid determination of DNA sequences and, by inference, the amino acid sequences of proteins from structural genes. The number of such sequences is now increasing almost exponentially, but by themselves these sequences tell little more about the biology of the system than a New York City telephone directory tells about the function and marvels of that city. 

The basic structural unit of these two-sheet p helix structures contains 18 amino acids, three in each p strand and six in each loop. A specific amino acid sequence pattern identifies this unit namely a double repeat of a nine-residue consensus sequence Gly-Gly-X-Gly-X-Asp-X-U-X where X is any amino acid and U is large, hydrophobic and frequently leucine. The first six residues form the loop and the last three form a p strand with the side chain of U involved in the hydrophobic packing of the two p sheets. The loops are stabilized by calcium ions which bind to the Asp residue (Figure S.28). This sequence pattern can be used to search for possible two-sheet p structures in databases of amino acid sequences of proteins of unknown structure. 

The unique characteristic of each protein is the distinctive sequence of amino acid residues in its polypeptide chain(s). Indeed, it is the amino acid sequence of proteins that is encoded by the nucleotide sequence of DNA. This amino acid sequence, then, is a form of genetic information. By convention, the amino acid sequence is read from the N-terminal end of the polypeptide chain through to the C-terminal end. As an example, every molecule of ribonucle-... 

Single-base substitutions, which affect the amino acid sequence of proteins and lead to altered protein function, are the most frequent type of polymorphisms associated with many disease phenotypes as well as with variation in drag response... 

Lindley, H. (1956) A new synthetic substrate for trypsin and its application to the determination of the amino acid sequence of proteins. Nature (London) 178, 647. 

Most of the genetic information stored in the genome codes for the amino acid sequences of proteins. For these proteins to be expressed, a text in nucleic acid language therefore has to be translated into protein language. This is the origin of the use of the term translation to describe protein biosynthesis. The dictionary used for the translation is the genetic code. 

The knowledge of amino acid sequences of proteins may provide their three-dimensional structures if CD predictions are valid. However, the CD prediction for the secondary structure of a given protein, especially having (3-sheet structures, is not valid enough to probe the structure-function relationships in native proteins. We are awaiting the development of a more precise prediction method for secondary structures of proteins. 

Transfer (tRNA) and ribosomal RNA (rRNA) are transcribed and used in protein synthesising processes. Messenger RNA (mRNA) codes the amino-acid sequence of proteins and 95 per cent of the total DNA transcribed is used for this purpose. In prokaryotes a single mRNA molecule may code for a single polypetide or for two or more polypeptide chains. There is a triplet code for each amino-acid 300 ribonucleotides code for a 100 amino-acid sequence. Fig. 5.All shows the relationship between the nucleotide sequence on DNA and RNA and the amino-acid sequence of protein. 

Determination of Amino Acid Composition of Proteins Determination of Amino Acid Sequence of Proteins Chemical Synthesis of Peptides and Polypeptides... 

The amino acid sequences of proteins and nucleotide sequences of DNA can be retrieved from the integrated database retrieval systems Entrez (http //... 

The amino acid sequences can be searched and retrieved from the integrated retrieval sites such as Entrez (Schuler et al., 1996), SRS of EBI (http //srs.ebi.ac.uk/), and DDBJ (http //srs.ddbj.nig.ac.jp/index-e.html). From the Entrez home page (http //www.ncbi.nlm.nih.gov/Entrez), select Protein to open the protein search page. Follow the same procedure described for the Nucleotide sequence (Chapter 9) to retrieve amino acid sequences of proteins in two formats GenPept and fasta. The GenPept format is similar to the GenBank format with annotated information, reference(s), and features. The amino acid sequences of the EBI are derived from the SWISS-PROT database. The retrieval system of the DDBJ consists of PIR, SWISS-PROT, and DAD, which returns sequences in the GenPept format. 

Phylogenetic analysis is the means of inferring or estimating evolutionary relationships. Nucleotide sequences of DNA or RNA and amino acid sequences of proteins are the most popular data used to construct phylogenetic trees. Methods of phylogenetic analysis using sequence data are introduced and performed with a software package, PHYLIP locally and online. 

This interaction pattern is also reflected in the amino acid sequences of proteins such as tropomyosin, which can be shown to be composed of a basic seven-residue sequence that is repeated 40 times without interruption (Hodges et al., 1972 McLachlan and Stewart, 1975). Hydrophobic residues almost invariably occupy the second and fifth positions of the heptad and are presumably directed toward the major axis of the superhelix, where they serve to stabilize the structure by hydrophobic effects (Fig. 15). The charged side chains are also nonrandomly distributed and are believed to form interhelical ion pairs that further stabilize the structure (Talbot and Hodges, 1982). 

Determining amino acid sequences of proteins by classic procedures is a tedious process. In addition, proteins are often insoluble (e.g., membrane proteins) or cannot be easily purified. However, if the protein s gene, mRNA, or cDNA of the mRNA are available, the amino acid sequence of the protein can be determined from nucleotide sequences rapidly and unambiguously using the universal genetic code. In this code, each amino acid in correlated with one or more nucleotide triplets in mRNA (see Chapter 12). mRNA is "transcribed" from DNA and can be used to synthesize complementary (cDNA) via an enzyme called reverse transcriptase. In many cases it is easier to isolate the mRNA or cDNA of a protein than the protein itself. 

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