Circular dichroism spectroscopy of three tyrosine-to-phenylalanine substitutions of fd gene 5 protein.
Circular dichroism spectroscopy was used to study mutants of phage fd gene 5 protein (Y26F, Y34F, and Y41F) in which three of the five tyrosines, Tyr-26, Tyr-34, and Tyr-41, were individually substituted with phenylalanine. The tyrosine 229 nm CD bands of the wild type, Y26F, and Y41F gene 5 proteins decreased in magnitude during complex formation with either fd ssDNA or poly[d(A)]. However, the Y34F gene 5 protein showed no decrease in the 229 nm band during titrations of these nucleic acids. This suggested that Tyr-34 of the wild type gene 5 protein dominated the 229 nm CD changes upon binding to single-stranded DNA. Titrations of poly[d(A)] or fd ssDNA with wild type, Y26F, Y34F, or Y41F gene 5 proteins resulted in perturbations of the nucleic acid near-UV CD bands, specific for the particular nucleic acid, but similar for all four proteins (in 2 mM sodium phosphate buffer, pH 7.0). For both nucleic acids, the addition of protein beyond a certain [protein monomer]/[nucleotide] ratio (0.25 for poly[d(A)] or 0.33 for fd ssDNA) resulted in a partial reversal of the CD change of the nucleic acid. These data are interpreted to mean that, in addition to the two well-known n = 4 and n = 3 stoichiometric modes of binding, there is a third mode of binding in which the nucleic acid is in limited contact with the protein. As shown by salt dissociation studies of complexes with poly[d(A)], the binding affinities, K omega, of the proteins were in the order: wild type > Y26F >> Y34F > or = Y41F (for the n = 4 binding mode in 0.1-0.2 M NaCl). Our data indicate that Tyr-34 plays a more important role in forming a complex with ssDNA than is apparent in current models of the g5p.ssDNA complex. We suggest that the hydroxyl moieties of Tyr-34 and Tyr-41 are both somehow involved in stabilizing the interface of bound protein dimers.
Mark
B L
BL
Program in Molecular and Cell Biology, University of Texas at Dallas, Richardson 75083-0688, USA.
Terwilliger
T C
TC
Vaughan
M R
MR
Gray
D M
DM
D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D009696:Nucleic Acids / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry*; D011485:Protein Binding; D012492:Salts; D014443:Tyrosine / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009696:Nucleic Acids; D012492:Salts; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13; D014443:Tyrosine; D010649:Phenylalanine