Primary structure of the bacteriophage T4 DNA helix-destabilizing protein

Abstract

The amino acid sequence of the single-stranded DNA-binding protein encoded by gene 32 of bacteriophage T4 has been determined by manual and automated sequencing of peptides derived from cyanogen bromide cleavage and digestion with trypsin, chymotrypsin, and staphylococcal protease. Tryptic digestion of citraconylated or succinylated gene 32 protein yields five peptides containing 4, 27, 42, 65, and 163 residues, respectively, which can be separated by Sephadex chromatography. Each of these tryptic peptides was subjected to automated sequencing and, if necessary, more extensive cleavage. The gene 32 protein contains 301 amino acids and has a molecular weight of 33,487. Based on its primary structure, the gene 32 protein is predicted to contain 36% alpha helix, 18% beta sheet, and 46% random coil. The native protein can be specifically cleaved at lysine 21 and 253 by limited trypsin digestion. Previous studies have shown that the "B" region (residues 1 to 21) is essential for cooperative binding to single-stranded DNA. The "A" region (residues 254 to 301) has been implicated in controlling the helix-destabilizing "activity" of gene 32 protein and in interacting with other T4 DNA replication proteins. The "A" region has a net charge of -10 and, in addition, contains two unusual stretches of 4 serine residues separated by glycine 284. The region between positions 72 and 116 contains 6 of the 8 tyrosine residues in the protein and may be important for DNA binding.