Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface

Abstract

E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy.

Cognate DNA Stabilizes the Tumor Suppressor p53 and Prevents Misfolding and Aggregation

“…To evaluate the contributions of DNA binding to p53C stability, we investigated its denaturation profile by measuring high-pressure effects on p53C conformation. Hydrostatic pressure has been widely used to assess the dissociation and denaturation of folded proteins, protein−nucleic acid complexes, and amyloid aggregates − . In Figure , we measured the pressure effects on p53C in the absence of DNA (circles) or in the presence of consensus (squares) or nonspecific (poly(GC)) (triangles) sequences.…”

IHF-binding sites inhibit DNA loop formation and transcription initiation

“…The reason why IHF-binding site itself inhibits the DNA loop formation is not clear. One possibility is that the IHF-binding site has a strong distortion or writhe [based on the reports that DNA deformation energy is low for IHF-binding sites ( 30 , 31 )], which guides its flanking DNA fragments into different planes, potentially reducing enhancer–promoter communication.…”

Hydrostatic Pressure Effects on the Fluorescence and FRET Behavior of Cy3-Labeled Phycocyanin System

“…Therefore, FRET has been used as a powerful tool in many biological fields such as biosensors, bioimaging, protein folding and unfolding, biomolecular interactions, and so on. − It is also potential to applying FRET to high hydrostatic pressure studies on biomacromolecules. In some articles, the RNA tertiary structure changes, protein–DNA binding affinity, and protein denaturation and aggregation have been explored through FRET. − Herein, we construct a FRET system between Cy3 label and C-phycocyanin and try to explore how pressure affects the fluorescence of the donor chromophore and acceptor protein, and especially on the FRET behavior between them in a Cy3-labeled C-phycocyanin (Cy3/C-PC) system. In addition, understanding the pressure effect on FRET behavior will highlight the investigation on the pressure-induced protein conformational changes, stability, function, and weak interaction between proteins, and so on.…”