Bioorganic models for transmembrane proteins

描述(申请人提供)：自从安芬森证明蛋白质的序列决定其结构以来，人们以越来越高的时间和空间分辨率探索了折叠的能量学和动力学。然而，几乎所有这些研究都致力于水溶性蛋白，而我们对膜蛋白的了解还处于初级阶段。在这里，我们使用蛋白质设计来测试和提炼我们对膜蛋白折叠的理解。模型肽被设计成在胶束和磷脂双层中缔合成螺旋束。由于这些体系的展开状态被很好地定义(单体螺旋)，并且可以通过测量单体-Nmer平衡来监测折叠的能量，从而简化了对这些体系的研究。在目标1和2中，我们测试了不同作用力的重要性和相对能量贡献，例如范德华堆积和极性相互作用。这些研究不仅将加深我们对蛋白质折叠的理解，而且也为理解跨膜螺旋结合的过程提供了洞察力和方法，而跨膜螺旋结合通常在信号转导中起着至关重要的作用。作为我们对膜蛋白折叠理解的结果，我们开始设计2种膜蛋白的水溶版本，磷蛋白和KCSA钾通道。这些研究有望为药物和生物物理研究提供一种获得大量水溶性膜蛋白的新方法。我们的具体目标如下：目标1.我们将研究设计的和天然的跨膜多肽的结合，并利用这些系统来了解导致跨膜螺旋折叠和结合的特征。目的2.我们将探索位于水基、头基和膜内部的蛋白质区域如何通过设计包含跨膜和水溶性折叠基序的螺旋束来决定蛋白质的折叠。目的3我们将对磷蛋白和KCSA的水溶性版本进行结构表征。目的：4.我们将开发跨膜蛋白结构预测和设计的计算方法。

DESCRIPTION (provided by applicant): Since Anfinsen's demonstration that the sequence of a protein dictates its structure, the energetics and kinetics of folding have been explored with ever-greater temporal and spatial resolution. However, almost all of these studies have been devoted to water-soluble proteins, and our understanding of membrane proteins is in its infancy. Here, we use protein design to test and refine our understanding of membrane protein folding. Model peptides are designed to associate into helical bundles in micelles and phospholipid bilayers. The study of these systems is simplified by the fact that the unfolded state is well defined (the monomeric helices), and the energetics of folding can be monitored by measuring the monomer-nmer equilibrium. In aims 1 & 2, we test the importance and relative energetic contributions of disparate forces such as van der Waals packing versus polar interactions. These studies will not only further our understanding of protein folding, but also provide insight and methods to understand the process of transmembrane helix association, which often plays an essential role in signal transduction. As an outgrowth of our understanding of the folding of membrane proteins, we are beginning to design water-soluble versions of 2 membrane proteins, phospholamban and the KcsA potassium channel. These studies promise to provide a new method for obtaining large quantities of water-soluble versions of membrane proteins for pharmaceutical and biophysical studies. Our specific aims are as follows: Aim 1. We will study the association of designed and natural transmembrane peptides, and use these systems to understand the features leading to the folding and association of transmembrane helices. Aim 2. We will explore how regions of a protein located in the aqueous, headgroup, and membrane interior cooperate to dictate the fold of a protein by designing helical bundles that incorporate both transmembrane as well as water-soluble folding motifs. Aim 3 We will structurally characterize water-soluble versions of phospholamban and KcsA. Aim4. We will develop computational methods for transmembrane protein structure prediction & design.