Ultrafast Fluorescence Decay Profiles Reveal Differential Unstacking of 2-Aminopurine from Neighboring Bases in Single-Stranded DNA-Binding Protein Subsites

Abstract

Gene 5 protein (g5p) is a dimeric single-stranded DNA-binding protein encoded by Ff strains of Escherichia coli bacteriophages. The 2-fold rotationally symmetric binding sites of a g5p dimer each bind to four nucleotides, and the dimers bind with high cooperativity to saturate antiparallel single-stranded DNA (ssDNA) strands. Ultrafast time-resolved fluorescence spectroscopies were used to investigate the conformational heterogeneity and dynamics of fluorescent 2-aminopurine (2AP) labels sequestered by bound g5p. The 2AP labels were positioned within the noncomplementary antiparallel tail sequences of d(AC)(8) or d(AC)(9) of hairpin constructs so that each fluorescent label could probe a different subsite location within the DNA-binding site of g5p. Circular dichroism and isothermal calorimetric titrations yielded binding stoichiometries of approximately six dimers per oligomer hairpin when tails were of these lengths. Mobility shift assays demonstrated the formation of a single type of g5p-saturated complex. Femtosecond time-resolved fluorescence spectroscopy showed that the 2AP in the free (non-protein-bound) DNAs had similar heterogeneous distributions of conformations. However, there were significant changes, dominated by a large increase in the population of unstacked bases from ~22 to 59-68%, depending on their subsite locations, when the oligomers were saturated with g5p. Anisotropy data indicated that 2AP in the bound state was less flexible than in the free oligomer. A control oligomer was labeled with 2AP in the loop of the hairpin and showed no significant change in its base stacking upon g5p binding. A proposed model summarizes the data.

Time-Resolved Down-Conversion of 2-Aminopurine in a DNA Hairpin: Fluorescence Anisotropy and Solvent Effects

“…Given this time scale, it is attributed to motions of the hairpin loop. The faster 0.1-ns component is most probably due to locally restricted motion of 2-AP and represents the limiting time scale between static and dynamic fluorescence quenching, as was pointed out in recent studies [1,3,5].…”

“…The peak of 2-AP fluorescence in aqueous solutions is at 371 nm, with a single decay constant of 10.4 ns. When incorporated to DNA, 2-AP fluorescence is extensively quenched [1][2][3].…”

Nucleotides sequestered at different subsite loci within DNA‐binding pockets of two OB‐fold single‐stranded DNA‐binding proteins are unstacked to different extents

“…By measuring the fluorescence decay profiles of 2AP in these oligomers, free or when complexed with g5p, we have explored the change in 2AP conformational heterogeneity during binding and how this change in heterogeneity is affected by binding at different subsite loci within the g5p protein's binding site. In previous work, we found that g5p binding to 2AP in the sequence context of C‐2AP‐C caused the probe to significantly unstack from neighboring cytosines at two different sub‐binding‐site locations . In the present work, for the first time, we have examined the influence of g5p binding on the local conformation of 2AP in the context of A‐2AP‐A and found that 2AP experienced an even greater degree of apparent unstacking from neighboring adenines compared with that when the 2AP was between neighboring cytosines.…”

Probing of Nucleic Acid Structures, Dynamics, and Interactions With Environment-Sensitive Fluorescent Labels

“…2AP was also employed as a lifetime probe for studying the kinetics and thermodynamics of conformational changes of DNA or RNA upon binding to proteins (Nguyen et al, 2011). In particular, a time-resolved technique was used to monitor flipping of 2AP within complexes of double-stranded DNA with the DNA methyltransferases M.HhaI and M.TaqI.…”