Control of Bacterial Nucleoid Structure

细菌核结构的控制

• 批准号: 8036899
• 负责人: JOHN W SHRIVER
• 金额: $ 44.13万
• 依托单位: UNIVERSITY OF ALABAMA IN HUNTSVILLE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8036899
• 关键词: Control; Bacterial; Nucleoid; Structure

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.

描述（由申请人提供）：我们的长期目标是准确描述 IHF、HU 和相关蛋白在控制细菌核结构和基因表达中的作用。这些蛋白质的变化通过核结构和基因调控的变化与代谢、生长和毒力的变化相关。在本提案中，我们重点关注 IHF 和相关蛋白质的结构、溶液状态以及 DNA 结合之间的关系。尽管 IHF 通常被视为序列特异性 DNA 结合蛋白，而 HU 是非序列特异性同系物，但越来越清楚的是，这种观点过于简单化。我们对这些蛋白质的结构了解很多，无论是在溶液中游离的还是与 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质对 DNA 结合的影响的了解是有限的且令人困惑的。特别是，IHF 的 DNA 结合能量非常出色，目前认为这是由于大量“掩蔽”表面盐桥控制了 DNA 结合和弯曲的盐和温度依赖性。一些工作人员认为这是 DNA 包裹表面的“特征”，并且有人提出它对于许多 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 结构和解决方案行为的重要属性。我们假设 IHF 不寻常的 DNA 结合能量和 DNA 结合的盐/温度依赖性与 IHF 的边缘稳定性和内在紊乱以及伴随结合的诱导折叠有关。为了测试这一点并为未来研究涉及 IHF 和相关蛋白质的高阶结构的组装和控制奠定坚实的基础，我们建议使用 NMR 搜索这些蛋白质中的表面盐桥，并对蛋白质溶液进行系统表征使用 ITC、CD、DSC 和分析超速离心分析特性及其与 DNA 结合的联系。为了研究该提议的普遍性，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 溶液特性和状态的描述，这对于严格正确地理解 IHF/HU 蛋白家族特异性和非特异性结合的控制和相对亲和力至关重要。 公共健康相关性：这项工作的结果将为一系列蛋白质的溶液状态提供准确、定量的描述，这些蛋白质在细菌生长和人类疾病中重要的许多蛋白质-DNA 相互作用中发挥着核心作用。这项工作是了解这些蛋白质通过 DNA 包装和基因调控的变化来控制细菌基因表达、代谢、生长和毒力的作用的必要的第一步。