Circular Dichroism Spectroscopy of Three Tyrosine-to-Phenylalanine Substitutions of fd Gene 5 Protein

Abstract

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.

Binding and reversible denaturation of double‐stranded DNA by Ff gene 5 protein

“…As more protein was added, there appeared to be a second break point at a [P]/[N] molar ratio of about 0.33. These break points seemed to correspond to the well‐documented n = 4 and 3 binding modes of g5p for ssDNA, in which one g5p monomer binds four or three nucleotides 29, 30. However, these break points in the titrations of poly[d(A) · d(T)] should not be considered as true binding modes, but rather as apparent binding modes, because it seems unlikely that successive g5p dimers could be arrayed so that each monomer would bind with equivalent contacts to the backbone of a right‐handed dsDNA.…”

“… b For complexes with poly[d(A)], the data for the WT, Y41F, Y41H, and Y56H proteins were from Mou et al30; the data for Y26F, Y34F, and additional data for Y41F were from Mark et al29; and the value for Y61F was from Thompson et al14 …”

“…The wild‐type and mutant (Y26F, Y34F, Y41H, Y56H, and Y61F) Ff gene 5 proteins were isolated from E. coli K561 cells containing a plasmid encoding gene 5, according to previously published procedures 19, 29–31. The protein was over 99% pure, as determined by quantitation of Coomassie Blue stained bands on 18% SDS‐PAGE gels.…”

“…The protein was over 99% pure, as determined by quantitation of Coomassie Blue stained bands on 18% SDS‐PAGE gels. The protein was finally dialyzed into 2 m M Na + (phosphate buffer, pH 7.0) and the concentrations were determined by UV absorption measurements using molar extinction coefficients [ϵ (276)] of 7074 and 5660 M −1 cm −1 for the wild‐type and mutant proteins, respectively 28–30…”

“…It has a large positive 229‐nm CD band that comprises contributions from all five tyrosine residues of the monomer 14, 28–30. When the protein forms a complex with ssDNA, the tyrosine 229‐nm CD band decreases in magnitude by up to 49% because of the perturbation of Tyr34 at the interface of adjacent protein dimers in the complex 14, 29, 30. An unresolved question has been whether Tyr34 is similarly perturbed when the g5p binds to dsDNA.…”