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

项目概要/摘要 该提案结合了我的两项 NIGMS 资助。我们对甲型流感 M2 质子通道的研究 病毒（GM56423）目前主要研究质子通过通道运动的机制。 M2 也是 金刚烷胺类流感药物的靶标，大多数甲型流感病毒分离株现在都是金刚烷胺- 抵抗的。 M2在各种功能状态下的晶体结构将以非常高分辨率解析 使用 X 射线自由电子激光能够在室温下确定结构。平行线， 合作研究使用单分子测量和 2DIR 来探测动力学。非常高分辨率 与M2金刚烷胺抗性突变体复合的一系列小分子抑制剂的结构是 正在确定，以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。我们解决膜中序列特异性识别的问题。各种 存在用于设计或选择识别水溶性抗体和其他试剂的方法 蛋白质区域。然而，针对跨膜 (TM) 区域的配套方法并不适用。 一般可用。因此，我们正在开发针对目标肽的计算设计方法 TM 螺旋以序列特异性方式形成，重点关注 EGF 受体（与 Natalia Jura 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，我们设计了模型蛋白质，使用质子梯度来驱动过渡金属离子的运输 向上一个梯度。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 允许设计磷酸盐转运蛋白和脂质翻转酶。我们建议继续进行设计工作 对二铁蛋白进行建模，以确定蛋白质如何调整这些辅因子的特性来影响不同的 O2- 依赖的过程，例如底物氧化和自由基形成。我们正在设计水和 蛋白质的膜溶性版本；通过改变配体和水的身份和几何形状 该中心的可访问性决定了这些参数如何定义反应性。 我们正在研究细菌组氨酸激酶传递构象的机制 通过多结构域 TM 蛋白传递信息。 HK 被细菌广泛用来感知和响应 不同的环境因素，例如营养素或有毒物质。各种截断晶体结构 HK的域名已经解决。然而，HK膜还没有高分辨率的结构—— 跨域或全长 HK，其信号机制仍存在争议。通过整合 来自不同实验技术和 HK 功能测量的结构信息，我们寻求 阐明 HK 中的信号传导机制。 59