Mapping DNA conformations and interactions within the binding cleft of bacteriophage T4 single-stranded DNA binding protein (gp32) at single nucleotide resolution

Abstract

In this study, we use single-stranded DNA (oligo-dT) lattices that have been position-specifically labeled with monomer or dimer 2-aminopurine (2-AP) probes to map the local interactions of the DNA bases with the nucleic acid binding cleft of gp32, the single-stranded binding (ssb) protein of bacteriophage T4. Three complementary spectroscopic approaches are used to characterize these local interactions of the probes with nearby nucleotide bases and amino acid residues at varying levels of effective protein binding cooperativity, as manipulated by changing lattice length. These include: (i) examining local quenching and enhancing effects on the fluorescence spectra of monomer 2-AP probes at each position within the cleft; (ii) using acrylamide as a dynamic-quenching additive to measure solvent access to monomer 2-AP probes at each ssDNA position; and (iii) employing circular dichroism spectra to characterize changes in exciton coupling within 2-AP dimer probes at specific ssDNA positions within the protein cleft. The results are interpreted in part by what we know about the topology of the binding cleft from crystallographic studies of the DNA binding domain of gp32 and provide additional insights into how gp32 can manipulate the ssDNA chain at various steps of DNA replication and other processes of genome expression.

Characterization of the T4 gp32–ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry

“…† These results indicate the ability of native MS to monitor the oligomerization of gp32 via binding to ssDNA as the length of the oligonucleotide increases, and are consistent with an approximately 6,7nucleotide footprint, as reported in previous gp32 studies. 10,78,79 These results likewise establish that native MS can distinguish the stoichiometries of native protein-DNA complexes and reveal the oligomerization of gp32 and its binding to ssDNA as a function of the DNA strand length.…”

A Spectroscopic Approach to Unravel the Local Conformations of a G-Quadruplex Using CD-Active Fluorescent Base Analogues

“…The quenching of fluorescence of base analogues in DNA by acrylamide depends on the solvent accessibility of fluorophores. Previously, we used this approach to acquire specifics of structural and dynamic aspects of DNA breathing near single-stranded–double-stranded junctions. ,, A higher quenching coefficient indicates more solvent accessibility, and we can monitor the differences in the solvent accessibility of the probe with variation in structural stability and conformational differences by this method. Acrylamide fluorescence quenching of 6MI residues in GQ was performed on 8Gm constructs to provide additional insight into the structure of the fluorescent G4 layer.…”

A Spectroscopic Approach to Unravel the Local Conformations of G-quadruplex Using CD-active Fluorescent Base Analogues

“…The quenching of fluorescence of base analogues in DNA by acrylamide depends on the solvent accessibility of fluorophores. Previously, we used this approach to acquire specifics of structural and dynamic aspects of DNA breathing near ss/ds junctions [29,41,46]. A higher quenching coefficient indicates more solvent accessibility, and we can monitor the differences in solvent accessibility of the probe with variation in structural stability and conformational differences by this method.…”