CIRCULAR-DICHROISM SPECTROSCOPY OF 3 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 tyros ines, Tyr-26, Tyr-34, and Tyr-41, were individually substituted with p henylalanine. 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 s howed no decrease in the 229 nm band during titrations of these nuclei c acids. This suggested that Tyr-34 of the wild type gene 5 protein do minated the 229 nm CD changes upon binding to single-stranded DNA. Tit rations 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 al l four proteins (in 2 mM sodium phosphate buffer, pH 7.0). For both nu cleic acids, the addition of protein beyond a certain [protein monomer ]/[nucleotide] ratio (0.25 for poly[d(A)] or 0.33 for fd ssDNA) result ed in a partial reversal of the CD change of the nucleic acid. These d ata 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 prote in. As shown by salt dissociation studies of complexes with poly[d(A)] , the binding affinites, K omega, of the proteins were in the order: w ild type > Y26F >> Y34F greater than or equal to Y41F (for the n = 4 b inding 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 i n current models of the g5p . ssDNA complex. We suggest that the hydro xyl moieties of Tyr-34 and Tyr-41 are both somehow involved in stabili zing the interface of bound protein dimers.