Control of Bacterial Nucleoid Structure

描述(由申请人提供)：我们的长期目标是对IHF、HU和相关蛋白在控制细菌类核结构和基因表达中的作用提供准确的描述。这些蛋白质的变化通过类核结构和基因调控的变化与新陈代谢、生长和毒力的变化联系在一起。在这项建议中，我们重点讨论结构、溶液状态和IHF和相关蛋白质与DNA结合的关系。虽然IHF通常被认为是序列特异的DNA结合蛋白，而HU是非序列特异的同系物，但越来越明显的是，这种观点过于简单化。我们对这些蛋白质的结构有很多了解，包括在溶液中游离的和与DNA结合的，但我们对它们在溶液中的行为以及溶剂条件和辅助蛋白对DNA结合的影响的了解是有限的和令人困惑的。特别是，IHF的DNA结合能是非同寻常的，目前人们认为这是由于大量被掩蔽的表面盐桥控制着DNA结合和弯曲的盐和温度依赖关系。这被一些工作人员认为是DNA包裹表面的“签名”，并已被提出对一些DNA结合蛋白具有普遍意义。我们认为这种解释是不正确的，因为它忽略了IHF结构和溶液行为的重要性质。我们假设IHF不寻常的DNA结合能和DNA结合的盐/温度依赖性与IHF的边缘稳定性和内在无序以及伴随结合的诱导折叠有关。为了验证这一点，并为未来涉及IHF和相关蛋白质的高阶结构的组装和控制的研究奠定坚实的基础，我们建议使用核磁共振技术寻找这些蛋白质中的表面盐桥，并使用ITC、CD、DSC和分析超速离心法系统地表征蛋白质溶液的性质及其与DNA结合的联系。为了研究这一建议的普遍性，我们将使用具有一系列稳定性和DNA结合亲和力的同源蛋白质的比较研究来明确定义结构和结合的联系。这项工作将提供IHF/HU溶液的性质和状态的描述，这对于严格正确地理解IHF/HU蛋白家族对特定和非特定结合的控制和相对亲和力是必不可少的。 公共卫生相关性：这项工作的结果将提供对蛋白质家族溶液状态的准确、定量的描述，这些蛋白质家族在许多蛋白质-DNA相互作用中发挥核心作用，这些蛋白质-DNA相互作用对细菌生长和人类疾病至关重要。这项工作是了解这些蛋白质通过改变DNA包装和基因调控来控制细菌基因表达、代谢、生长和毒力的必要的第一步。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.