Site-specific mutagenesis of T4 gene 32: the role of tyrosine residues in protein-nucleic acid interactions.
Bacteriophage T4 gene 32 encodes a single-stranded DNA (ssDNA) binding protein (gp32) required for T4 DNA replication, recombination, and repair. Previous physicochemical studies on gp32 and other ssDNA binding proteins have suggested that binding may involve hydrophobic interactions that result from the close approach of several aromatic amino acid side chains with the nucleic acid bases. In the case of gp32, five tyrosines and two phenylalanines have previously been implicated in gp32.ssDNA complex formation. Site-directed mutagenesis of T4 gene 32 was employed to produce a set of eight gp32 mutant proteins, each of which encoded a single substitution at one of the eight tyrosine residues within gp32. The mutant gp32 proteins were then subjected to physicochemical analysis to evaluate the role of each tyrosine residue in gp32 structure and function. Oligonucleotide binding studies suggest that tyrosine residues 84, 99, 106, 115, and 186 each contribute from 0.3 to 0.7 kcal/mol to ssDNA binding, which corresponds to 3-7% of the overall binding energy for gp32.ssDNA complex formation. Replacement of tyrosine residues 73 and 92 appears to lead to large structural changes that may be the result of disrupting the zinc binding subdomain within gp32.
Shamoo
Y
Y
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Ghosaini
L R
LR
Keating
K M
KM
Williams
K R
KR
Sturtevant
J M
JM
Konigsberg
W H
WH
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005814:Genes, Viral; D009154:Mutation; D009691:Nucleic Acid Denaturation; D011071:Poly T / Q000378:metabolism; D011067:Poly dA-dT / Q000378:metabolism; D011489:Protein Denaturation; D013604:T-Phages / Q000235:genetics*; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D015032:Zinc / Q000032:analysis
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D011071:Poly T; D011067:Poly dA-dT; D014443:Tyrosine; D014357:Trypsin; D015032:Zinc