Primary Structure

The backbone of polypeptide chains is described by two dihedral angles per residue: <p and y at the C" atom. In a peptide linkage -NH-CaH(R)-CO-NH-, the angles of -NH-C"-, -CT-CO-, and -CO-NH- are angles <p, v|i, and co, respectively (Fig. 1). The two broken lines in Figure 1 show the location of peptide linkages with an amino acid residue -NH-C"H(R)-CO- between these two linkages.

The genetic code involving the sequence of purine and pyrimidine bases in the DNA strand determines the se-

Figure 1. Polypeptide chain linking two peptide units. The chain is shown in a fully extended conformation with all ft, *|/„ and co, angles greater than 180°. Source: Ref. 15.

quence of amino acids along the protein chain. Each group of three bases along the messenger RNA strand specifies a particular amino acid, and the sequence of these triplet groups dictates the sequence of the amino acids in the proteins. The genetic code is, however, degenerate; that is, most amino acids are coded by more than one codon. Therefore, a nucleotide sequence cannot be derived from the colinear amino acid sequence. But an unknown amino acid sequence can be deduced from the colinear nucleotide sequences. Thus, protein sequence can be determined by analyzing the underlying nucleotide sequence.

Sequence Analysis

For peptide sequence analysis, solid-phase Edman degradation has been used. Lately, gas-phase microsequencing has become popular. First, a phenylthiocyanate or other similar isocyanate solution is added, and then lower al-kylamines, eg, triethylamine, in vapor are delivered by a stream of argon gas to convert the peptide sample on a porous support into a phenyl thiocarbamylated peptide. Then trifluoroacetic acid or another similar fluoride-containing organic acid in vapor form is delivered to liberate the phenylthiohydantion (pth) amino acid derivatives. The resultant pth-amino acids are extracted and analyzed by RP/HPLC. This cycle is repeated until it reaches the COOH terminus of the peptide chain (16). In the case of larger protein molecules, fragmentation to small peptides is needed since there are limits on the chain length that can be analyzed.

The advent of capillary electrophoresis in conjunction with nanoelectrospray mass spectrometry enabled the Ed-man sequencing of peptides and proteins using 5-10 pi-comoles of materials (17).

Recent developments in nucleic acid cloning and sequencing techniques have made the determination of the amino acid sequence of a protein straightforward, inexpensive, accurate, and rapid (18). For analysis of DNA sequences, two methods are widely used: the enzymatic di-deoxy method and the chemical method. The difference is primarily in the technique used to generate the ladder of oligonucleotides. In the enzymatic dideoxy sequencing method, a DNA polymerase is used to synthesize a labeled, complementary copy of a DNA template. In the chemical sequencing method, a labeled DNA strand is subjected to a set of base-specific chemical reagents. A set of radiolabeled single-stranded oligonucleotides is generated in four separate reactions, either enzymatically or chemically. In each of the four reactions, the oligonucleotides have one fixed and one end that terminates sequentially at each A, T, G, or C, respectively. The products of each reaction are fractionated by electrophoresis on adjacent lanes of a highresolution polyacrylamide gel. After autoradiography, the DNA sequence can be read directly from the gel.

Characteristic Properties of Amino Acids in Protein Structure

Each amino acid residue has its own characteristic property that cannot be replaced by another residue. All amino acid residues, except glycine, have characteristic side chains on the main chain linked through peptide linkage

(2). Glycine increases flexibility to the main chain because it has no side chain but with only two hydrogens. A polypeptide chain at a glycine residue has considerably more conformational freedom than at any other residue. Alanine is the smallest nonpolar residue that does not have much preference on whether it is located inside or on the surface of a protein molecule. The nonpolar side chains of valine, isoleucine, and leucine are branched, thus restricting internal flexibility. Phenylalanine has the largest nonpolar side chain. Because of the single methylene group on C/' as with the other two aromatic side chains (tyrosine and tryptophan), the side chain flexibility is restricted. Tyrosine has by far the most reactive side chain of the three aromatic residues because of its hydroxyl radical.

The side chain of proline is characteristic because the last atom of the side chain is bonded to the main chain N atom, forming a ring structure. This prevents the N atom from participating in hydrogen bonding and also provides a steric hindrance to the a-helical conformation (19). Consequently, proline has the smallest degree of conformational freedom of all the amino acids, and a polypeptide chain at a proline residue has appreciably less conformational freedom. This characteristic property of proline can be used for stabilizing protein structure; however, its location should be carefully chosen so that the new residue should neither create volume interferences nor destroy stabilizing noncovalent interactions (20). Proline introduction to the N-terminal end of active-site helix of Bacillus stearo-thermophilus neutral protease improved thermostability. The glycine residues on the TV-terminal side of proline residue relaxed the possible strain that resulted from proline introduction, thereby increasing molecular rigidity (21). Methionine has a rather flexible side chain with a sulfur in a thioether bond. This sulfur introduces an electrical dipole moment.

All the larger nonpolar residues, namely valine, isoleucine, leucine, phenylalanine, proline, tryptophan, and, to a lesser extent, methionine, are predominantly in the inside of protein molecules. Polar (uncharged) side chains form hydrogen bonds, for instance, serine and threonine have hydroxyl groups that form hydrogen bonds (1). Cysteine plays a special role by forming disulfide bridges between different parts of the main chain. A group can be activated in protein through specific hydrogen bonds, such as serine acting as a donor to an unprotonated imidazole group of a histidine. Such charge relay systems form an essential part of the active site of serine proteases. In both serine and threonine, the hydroxyl group can react with acids to form esters via enzyme-catalyzed reactions. Both are common sites for phosphorylation, fatty acid esterification, and glycosylation in proteins. The acid amides of asparagine and glutamine can also form hydrogen bonds. The amido groups function as hydrogen donors and the carbonyl groups as acceptors. The side chain of glutamine is more flexible than that of asparagine because of its extra methylene group. The polar hydroxyl group of tyrosine forms relatively strong hydrogen bonds.

Histidine has a heterocyclic aromatic side chain with a pK value of 6.0. In the physiological pH range, its imidazole ring can be either uncharged or charged. This chemical equilibrium is suitable for catalyzing reactions. This is one of the reasons why histidine is found in several of the active sites of enzymes. Aspartate and glutamate, usually located at the molecular surface, are negatively charged at physiological pH. Because of the short side chain, the car-boxyl groups of aspartate are relatively rigid. This may be a reason why the carboxyl groups of active sites of enzymes, eg, aspartyl proteinases, are mainly provided by aspartates and not glutamates.

Most of the positively charged lysine and arginine residues are also at the molecular surface. They are long and flexible and do not usually adopt a defined conformation. The surface net charges of these residues in counterbalance with surface hydrophobicity increase the solubility of globular proteins (22). Sometimes they participate in internal salt bridges or in catalysis. The e-amino group of lysyl residues, and to a lesser extent the guanidinium groups of arginyl residues, are the target of enzyme action, which either modifies the side chain or cleaves the peptide chain at the carboxyl end of lysyl and arginyl residues of substrates. The e-amino group of lysine is considered to be the second-most reactive group in proteins, second to the cysteine sulfhydryl group. The most famous reaction of the e-amino group of lysine is the formation of a Schiffbase by the so-called Maillard reaction with aldehydes.

The folding process of a polypeptide chain depends on the hydrophobicity of the side chains, because the formation of a hydrophobic core in the globule seems to be one of the essential driving forces in folding. The hydrophobicity of amino acid residues is dependent on water-accessible surface area and dipole content.

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