Ultrafast Fluorescence Decay Profiles Reveal Differential Unstacking of 2-Aminopurine Residues Positioned Within Binding Subsites of a Single-Stranded DNA Binding Protein

Abstract

DNAs bound by proteins are kinetically dynamic and exist in multiple conformations, including conformations that differ in stacking interactions. We have utilized ultrafast time resolved fluorescence spectroscopy to investigate changes in base stacking of single-stranded DNA (ssDNA) upon binding of the dimeric gene 5 protein (g5p) of fd, f1, and M13 strains of E. coli bacteriophages. DNA oligomer hairpins of 44 nucleotides were designed to have two antiparallel 16-nucleotide ssDNA tails for binding of a cluster of g5p dimers. Otherwise identical oligomers each contained a single fluorescent 2-aminopurine (2AP) base. Time domain fluorescence measurements showed that the label exists in an ensemble of conformations, including stacked, partially stacked, and unstacked species that are altered upon binding of the g5p. Two oligomers had 2AP labels at different subsite locations within one of the four-nucleotide DNA-binding sites of a given g5p monomer when the oligomers were saturated with g5p. These labels showed increases of the unstacked conformation from 22-24% for the free DNAs to 47-74% for the bound DNAs, where the extent of this increase was specific for the subsite location within the DNA-binding site of the g5p monomer. Time resolved anisotropy measurements indicated that the 2AP labels were in relatively inflexible conformations within the g5p-saturated oligomers.