Our long-term goal is to provide an accurate description of the role of IHF, HU, and associated proteins in controlling bacterial nucleoid structure and gene expression. Changes in these proteins are linked to changes in the metabolism, growth and virulence through changes in both nucleoid structure and gene regulation. In this proposal we focus on the relationship between structure, solution states, and DNA binding by IHF and related proteins. Although IHF is commonly viewed as a sequence-specific DNA binding protein, and HU a non-sequence specific homolog, it is becoming increasingly clear that this view is too simplistic. We know a great deal about the structures of these proteins, both free in solution and bound to DNA, but our knowledge of their behavior in solution and the effect of solvent conditions and accessory proteins on DNA binding is limited and perplexing. In particular, the DNA binding energetics of IHF are extraordinary and it is currently believed that this is due to a large number of """"""""masked"""""""" surface salt bridges which control the salt and temperature dependence of DNA binding and bending. This is thought by some workers to be a """"""""signature"""""""" for DNA wrapping surfaces and it has been proposed to be of general significance for a number of DNA binding proteins. We propose that this interpretation is incorrect because it ignores important properties of IHF structure and solution behavior. We hypothesize that the unusual DNA binding energetics of IHF and the salt/temperature dependence of DNA binding are linked to marginal stability and intrinsic disorder of IHF, and the induced folding that accompanies binding. In order to test this and build a firm foundation for future studies of the assembly and control of higher order structures involving IHF and related proteins, we propose a search for surface salt bridges in these proteins using NMR, along with a systematic characterization of protein solution properties and their linkage to DNA binding using ITC, CD, DSC, and analytical ultracentrifugation. To investigate the generality of this proposal, we will use comparative studies of homologous proteins with a range of stabilities and DNA binding affinities to unambiguously define the linkage of structure and binding. This work will provide a description of IHF/HU solution properties and states which are essential to providing a rigorously correct understanding of the control and relative affinities of specific and non-specific binding by the IHF/HU protein family.
The results of this work will provide an accurate, quantitative description of the solution states of a family of proteins which play a central role in many protein-DNA interactions important in bacterial growth and human disease. This work is a necessary first step to understanding the role of these proteins in controlling bacteria gene expression, metabolism, growth and virulence through changes in both DNA packaging and gene regulation.
