Role of Tyr-22 in the binding of Pf3 ssDNA binding protein to nucleic acids.
A Tyr-22-->Phe-22 (Y22F) mutant of the single-strand DNA binding protein (ssDBP) of the filamentous phage Pf3 was obtained by site-directed mutagenesis. An alignment of protein sequences indicates that Tyr-22 of the Pf3 ssDBP corresponds to Tyr-26 of the fd g5p, a tyrosine within the DNA-binding loop. The mutant Y22F Pf3 protein had a CD spectrum very similar to that of native, wild-type Pf3 ssDBP and could bind to both DNA and RNA polymers. In CD titrations of poly[r(A)], poly[r(C)], and Pf3 ssDNA with the Y22F mutant, the saturation endpoints remained the same as for titrations performed with wild-type Pf3 ssDBP, indicating that the mutant protein retained the same n = 2 mode of binding as the wild-type protein. However, a second stoichiometric mode of binding at a ratio of one protein monomer to about four nucleotides (n = 4) was observed for titrations of these nucleic acids with the Y22F mutant protein. Both proteins showed only an n = 2 mode of binding to poly[d(A)], poly[d(C)], and poly[d(T)] and only an n = 3 mode of binding to poly[r(U)]. Distinctly different CD spectral changes of the nucleic acid were observed in titrations of poly[d(A)] with the Y22F mutant and the wild-type protein. Therefore, the mutant and wild-type ssDBP interact differently with some nucleic acids, depending on the base and sugar composition, providing evidence that Tyr-22 is indeed in the DNA-binding loop and may be important in the sequence discrimination of the binding of the Pf3 ssDBP.
Powell
M D
MD
Program in Molecular and Cell Biology, University of Texas at Dallas, Richardson 75083-0688, USA.
Gray
D M
DM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010649:Phenylalanine; D010957:Plasmids; D017354:Point Mutation; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011131:Polyribonucleotides / Q000378:metabolism; D011487:Protein Conformation; D017105:Pseudomonas Phages / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014443:Tyrosine
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011089:Polydeoxyribonucleotides; D011131:Polyribonucleotides; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine