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

项目总结/摘要 这份提案结合了我的两个NIGMS赠款。我们对甲型流感M2质子通道的研究 病毒（GM 56423）目前主要关注质子通过该通道的运动机制。M2也是 金刚烷胺类流感药物的靶点，大多数甲型流感病毒分离株现在都是金刚烷胺- 抵抗M2在各种功能状态下的晶体结构将以非常高的分辨率得到解决 使用X射线自由电子激光器，使结构确定在室温下。平行， 协作研究使用单分子测量和2 HNMR来探测动力学。非常高分辨率 与M2的金刚烷胺抗性突变体复合的一系列小分子抑制剂的结构， 正在确定，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（GM 54616）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。我们解决的问题，在膜上的序列特异性识别。各种 存在用于设计或选择抗体和其它试剂的方法 蛋白质的区域。然而，靶向跨膜（TM）区域的伴随方法并不 一般可用。因此，我们正在开发用于肽的计算设计的方法， 以序列特异性方式研究TM螺旋，重点关注EGF受体（与娜塔莉亚尤拉合作）， 整合素（与A. Orr）。阐明质子偶联转运蛋白 功能，我们已经设计了模型蛋白质，使用质子梯度来驱动过渡金属离子的运输 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。我们建议继续设计 模型二铁蛋白，以确定蛋白质如何调节这些辅因子的特性，以影响不同的O2- 依赖的过程，例如衬底氧化和自由基形成。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， 中心的可访问性，以确定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蛋白的信息。HK被细菌广泛用于感知和响应 不同的环境线索，如营养物质或有毒物质。各种截顶晶体结构 HK的域已被解决。然而，HK膜没有高分辨率的结构- 跨域或全长HK，它们的信号传导机制是一个有争议的问题。通过整合 我们试图从不同的实验技术和功能测量的HK的结构信息， 阐明HK信号转导机制。 59

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