Deciphering the relationship between structure, dynamics and function in helical bundle proteins

解读螺旋束蛋白的结构、动力学和功能之间的关系

• 批准号: 9977222
• 负责人: WILLIAM DEGRADO
• 金额: $ 71.19万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977222
• 关键词: Address; Affect; Amantadine; Amantadine resistance; Antibodies; Bacteria; Collaborations; Companions; Complex; Computing Methodologies; Coupled; Crystallization; Cues; Drug Design; Environment; Epidermal Growth Factor Receptor; Geometry; Grant; Influenza; Influenza A virus; Inorganic Phosphate Transporter; Integral Membrane Protein; Integrins; Ions; Length; Ligands; Lipids; Measurement; Membrane; Membrane Proteins; Methods; Modeling; Molecular Conformation; Movement; National Institute of General Medical Sciences; Nutrient; Peptides; Pharmaceutical Preparations; Process; Property; Protein Engineering; Protein Region; Proteins; Protons; Reagent; Resolution; Series; Signal Transduction; Structure; Techniques; Temperature; Testing; Transition Elements; Transmembrane Domain; Water; Work; base; cofactor; design; model design; mutant; oxidation; protein structure function; protein-histidine kinase; single molecule; small molecule inhibitor; x-ray free-electron laser

Project Summary/Abstract This proposal combines my two NIGMS grants. Our work on the M2 proton channel from influenza A virus (GM56423) currently focuses on the mechanism of proton movement through the channel. M2 is also the target of the amantadine class of influenza drugs, and most isolates of influenza A virus are now amantadine- resistant. Crystallographic structures of M2 in various functional states will be solved at very high resolution using the X-ray free electron laser to enable structure determination at room temperature. Parallel, collaborative studies use single-molecule measurement and 2DIR to probe dynamics. Very high-resolution structures of a series of small molecule inhibitors in complex with amantadine-resistant mutants of M2 are being determined, to enable our collaborators to conduct structure-based drug design. De novo protein design (GM54616) provides a means to test and refine our understanding of protein structure and function. We address questions of sequence-specific recognition in membranes. A variety of methods exist for the design or selection of antibodies and other reagents that recognize the water-soluble regions of proteins. However, companion methods for targeting Transmembrane (TM) regions are not generally available. Therefore, we are developing methods for the computational design of peptides that target TM helices in a sequence-specific manner, focusing on EGF receptors (collaboration with Natalia Jura) and integrins (collaboration with A. Orr). To elucidate the mechanisms by which proton-coupled transporters function, we have designed model proteins that use proton gradients to drive transport of transition metal ions up a gradient. We are increasing the efficiency of these minimal models and also expanding our methods to allow design of phosphate transporters and lipid flippases. We propose to continue work on the design of model diiron proteins to determine how a protein tunes the properties of these cofactors to affect diverse O2- dependent processes such as substrate oxidation and radical formation. We are designing water and membrane-soluble versions of the protein; by varying the identity and geometry of ligands and the water- accessibility of the center to determine how these parameters they define reactivity. We are studying the mechanisms by which bacterial histidine kinases transmit conformational information through multi-domain TM proteins. HKs are widely used by bacteria to sense and respond to diverse environmental cues such as nutrients or noxious substances. Crystal structures of various truncated domains of HKs have been solved. However, there are no high-resolution structures for HK membrane- spanning domains or full-length HKs, and their signaling mechanism is a matter of debate. By integrating structural information from diverse experimental techniques and functional measurements of HKs we seek to elucidate the mechanism of signaling in HKs. 59

项目摘要/摘要 该提案结合了我的两个纽约赠款。我们在流感A的M2质子通道上的工作 病毒（GM56423）当前关注通过通道质子运动的机理。 M2也是 阿曼塔丁类药物类药物的靶标，以及大多数流感病毒的分离株，现在是amantadine- 抵抗的。 M2在各种功能状态下的晶体学结构将以非常高的分辨率解决 使用X射线无电子激光器在室温下实现结构测定。平行线， 协作研究使用单分子测量和2DIR进行探测动力学。非常高分辨率 与M2的抗肿瘤抗性突变体复合物中一系列小分子抑制剂的结构 确定，使我们的合作者能够进行基于结构的药物设计。 从头蛋白质设计（GM54616）提供了一种测试和完善我们对蛋白质的理解的方法 结构和功能。我们解决了膜中序列识别的问题。各种各样 存在抗体和其他识别水溶性抗体和其他试剂的方法的方法 蛋白质区域。但是，靶向跨膜（TM）区域的伴侣方法不是 通常可用。因此，我们正在开发针对目标的肽的计算设计方法 以特定于序列的方式进行TM螺旋，重点是EGF受体（与Natalia Jura的合作）和 整合素（与A. ORR合作）。阐明质子耦合转运蛋白的机制 功能，我们设计了使用质子梯度驱动过渡金属离子传输的型号蛋白质 提高梯度。我们正在提高这些最小模型的效率，并将我们的方法扩展到 允许设计磷酸转运蛋白和脂质爆破。我们建议继续从事设计 模拟二氮蛋白，以确定蛋白质如何调节这些辅因子的特性，以影响多种O2-- 依赖性过程，例如底物氧化和自由基形成。我们正在设计水和 蛋白质的膜溶剂版本；通过改变配体的身份和几何形状和水 - 中心的可访问性确定它们如何定义反应性。 我们正在研究细菌组氨酸激酶传递构象的机制 通过多域TM蛋白的信息。细菌广泛使用HK来感知和反应 养分或有害物质等各种环境线索。各种截短的晶体结构 HK的域已解决。但是，没有用于HK膜的高分辨率结构 跨越域或全长HK，它们的信号传导机制是争论的问题。通过集成 来自不同实验技术和HK的功能测量的结构信息，我们寻求 阐明HKS中信号传导的机制。 59