Circular dichroism and fluorescence analysis of the interaction of Pf1 gene 5 protein with poly(dT)

Abstract

Circular dichroism (c.d.) and fluorescence spectroscopy have been used to investigate the interaction of the gene 5 protein of the filamentous bacteriophage Pfl with single-stranded DNA. The c.d. spectrum of the Pfl gene 5 protein is consistent with the absence of any significant α-helical content. The negative c.d. peak in the region of 210 nm, which arises from the protein, is diminished in the complex with poly(dT). Likewise, the c.d. peak at 265 nm arising from the poly(dT) decreases when the Pfl gene 5 protein is bound, c.d. titrations of poly(dT) with Pfl gene 5 protein indicate strong binding with a stoichiometry ( n) of four nucleotides per protein subunit. In contrast, when the titrations were done using fluorescence anisotropy or fluorescence spectral shifts to follow binding, apparent stoichiometries between n = 2 and n = 4 were observed, often in the same experiment, depending on precise conditions. The results are interpreted in terms of two distinct modes of binding, in which either one or two subunits of the protein dimer are bound to the polynucleotide lattice, but still retaining the same local interaction with the DNA, with each binding site covering four nucleotides. The apparent stoichiometry of 2 results from the interaction of only one subunit of the dimer with the nucleic acid lattice, when protein is in excess. The second, unfilled, subunit of the dimer is nevertheless incorporated into the complex, resulting in the maximum possible fluorescence change when only half the sites are filled, since the fluorescence properties of the complex arise from protein-protein contacts associated with co-operative binding to the lattice. Further experiments in which the order of addition of components is changed, and the concentration of MgCl 2 is varied, show that both of these factors are important in determining the dominant binding mode. In the absence of salt, dissociation and redistribution of the polynucleotide can occur following the addition of excess protein. This transition is suppressed in the presence of >3 m m-MgCl 2.