1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis.
Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005798:Genes, Bacterial; D006639:Histidine; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009154:Mutation; D010649:Phenylalanine; D014364:Tryptophan; D014443:Tyrosine
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine; D006859:Hydrogen; D014364:Tryptophan