The Covalent Structure of Proteins:- Amino Acid Sequences Can Also Be Deduced by Other Methods
The approach outlined above is not the only way to determine amino acid sequences. New methods based on mass spectrometry permit the sequencing of short polypeptides (20 to 30 amino acid residues) in just a few minutes (Box 3–2). In addition, with the develop ment of rapid DNA sequencing methods (Chapter 8), the elucidation of the genetic code (Chapter 27), and the development of techniques for isolating genes (Chapter 9), researchers can deduce the sequence of a polypeptide by determining the sequence of nucleotides in the gene that codes for it (Fig. 3–28). The techniques used to determine protein and DNA sequences are com plementary. When the gene is available, sequencing the DNA can be faster and more accurate than sequencing the protein. Most proteins are now sequenced in this in direct way. If the gene has not been isolated, direct sequencing of peptides is necessary, and this can provide information (the location of disulfide bonds, for example) not available in a DNA sequence. In addition, a knowledge of the amino acid sequence of even a part of a polypeptide can greatly facilitate the isolation of the corresponding gene (Chapter 9). The array of methods now available to analyze both proteins and nucleic acids is ushering in a new discpline of “whole cell biochemistry.” The complete sequence of an organism’s DNA, its genome, is now available for organisms ranging from viruses to bacteria to multicellular eukaryotes (see Table 1–4). Genes are being discovered by the millions, including many that en code proteins with no known function. To describe the entire protein complement encoded by an organism’s DNA, researchers have coined the term proteome. As described in Chapter 9, the new disciplines of genomics and proteomics are complementing work carried out on cellular intermediary metabolism and nucleic acid metabolism to provide a new and increasingly complete picture of biochemistry at the level of cells and even organisms.
FIGURE 3–27 Cleaving proteins and sequencing and ordering the peptide fragments. First, the amino acid composition and amino terminal residue of an intact sample are determined. Then any disulfide bonds are broken before fragmenting so that sequencing can proceed efficiently. In this example, there are only two Cys (C) residues and thus only one possibility for location of the disulfide bond. In polypep tides with three or more Cys residues, the position of disulfide bonds can be determined as described in the text. (The one-letter symbols for amino acids are given in Table 3–1.
FIGURE 3–28 Correspondence of DNA and amino acid sequences. Each amino acid is encoded by a specific sequence of three nucleotides in DNA.
