A novel approach to integral & anchored membrane protein structure & function

描述(申请人提供)：由结晶学和核磁共振(核磁共振)确定的高分辨率蛋白质结构模型的数量激增。然而，随着已知结构的数量接近50,000个，其中只有几百个是完整的膜蛋白。预计在不久的将来，结构生物学将在确定足够数量的独特蛋白质结构方面取得重大进展，以便能够仅根据序列预测任何结构。不幸的是，这一极具挑战性的目标由于完整的膜蛋白和可能令人惊讶的膜锚定蛋白所呈现的显著障碍而变得更加困难。这一建议试图摆脱这两类重要蛋白质带来的限制，并为利用溶液核磁共振方法在其结构和生物物理表征方面取得重大进展奠定基础。我们的方法是不同寻常的，而且还在不断涌现。它是基于我们早期的工作，使用反胶束封装来克服大容量可溶性蛋白质带来的缓慢翻滚问题。在这种方法中，感兴趣的蛋白质被包裹在反胶束颗粒的保护性水核中，整个组装件被溶解在低粘度的流体中，如液态乙烷。在低粘度流体中，反胶束粒子的翻滚速度比蛋白质在散装水中溶解的速度更快。这对控制现代三重共振实验效率的核磁共振弛豫特性提供了显著的改进。这种方法可以在100 kDa的可溶蛋白质上获得高性能的核磁共振谱，而不需要重氢或TROSY效应。在这里，我们建议采用这种方法来研究完整的和脂类锚定的外周膜蛋白。反胶束方法将用于表征KCSA钾通道跨膜段的结构。这将为研究形成通道的多肽链的动力学提供基础，这些多肽链被认为是离子选择性的重要因素。KCSA的研究将为测试该方法的通用性奠定基础，以表征完整的膜蛋白的结构。同时，我们将使用反胶束包裹来研究共价结合的脂类在蛋白质锚定到膜上的作用。我们将研究肉豆蔻酰化的HIV-1基质蛋白与磷酸肌苷PIP2结合的结构与功能的关系。这种相互作用已被认为是药物干预的有前途的靶点。经典的桃金娘花柱开关蛋白恢复素将被用来进一步定义该方法。对棕榈酰化蛋白BET3和UL11的类似研究也被提出，BET3是膜靶向复合体的组成部分，UL11是单纯疱疹病毒的被膜蛋白。蛋白质和通常嵌入在膜上的配体之间的相互作用也将被研究。这些研究应该建立反胶束增溶方法作为整体和脂质锚定的膜蛋白的结构和动力学研究的一般方法。与公共卫生相关：尽管完整的膜蛋白对人类生物学中的许多基本过程至关重要，并且代表了药物干预医学的大多数靶点，但在结构细节上表征它们仍然是极其困难的。在这里，我们将发展一种新的方法，通过核磁共振来确定完整的膜蛋白的结构。这一新方法还提供了膜锚定蛋白的独特视角，其中一些是抗病毒治疗的潜在靶点。

DESCRIPTION (provided by applicant): There has been an explosion in the number of high resolution structural models of proteins determined by crystallography and nuclear magnetic resonance (NMR). Yet as the number of known structures approaches 50,000 only a few hundred of those are of integral membrane proteins. The near future of structural biology is envisaged to make significant progress towards the determination of a sufficient number of unique protein structures to allow the prediction of any structure based on sequence alone. Unfortunately, this immensely challenging goal is made even more difficult by the significant barrier presented by integral membrane proteins and, perhaps surprisingly, by membrane anchored proteins. This proposal seeks to escape the restraints presented by these two important classes of proteins and set the stage for significant advances in their structural & biophysical characterization by solution NMR methods. Our approach is unusual and still emerging. It is based on our earlier work using reverse micelle encapsulation to defeat the slow tumbling problem presented by large soluble proteins. In that approach, the protein of interested is encapsulated within the protective aqueous core of a reverse micelle particle and the entire assembly is dissolved in a low viscosity fluid such as liquid ethane. In the low viscosity fluid, the reverse micelle particle tumbles faster than the protein dissolved in bulk water. This provides a significant improvement in the NMR relaxation properties governing the efficiency of the modern triple resonance experiments. The method allows high performance NMR spectra to be obtained on soluble proteins as large as 100 kDa without benefit of deuteration or the TROSY effect. Here we propose to adapt this approach to studies of integral and lipid-anchored peripheral membrane proteins. The reverse micelle method will be used structurally characterize the transmembrane segment of the Kcsa potassium channel. This will provide a foundation for the study of the dynamics of the polypeptide chain forming the channel, which are thought to be important to ion selectivity. Studies with Kcsa will set the stage for a test of the generality of the approach to structural characterization of integral membrane proteins. In parallel, we will employ reverse micelle encapsulation to study the role of covalently attached lipids in the anchoring of proteins to the membrane. We will investigate the structure-function relationships of the myristoylated HIV-1 matrix protein in its binding to the phosphoinositde PIP2. This interaction has been proposed to be promising target for pharmaceutical intervention. The classic myristyl-switch protein recoverin will be used to further define the approach. Analogous studies are proposed for the palmitoylated proteins BET3, a component of a membrane targeting complex, and UL11, the tegument protein of the herpes simplex-1 virus. Interactions between proteins and ligands normally embedded in the membrane will also be investigated. These studies should establish the reverse micelle solubilization method as general approach to structural and dynamic studies of integral and lipid-anchored membrane proteins. PUBLIC HEALTH RELEVANCE: Although integral membrane proteins are vital to many fundamental processes in human biology and represent the majority of targets for pharmaceutical intervention in medicine, it remains extremely difficult to characterize them in structural detail. Here we will develop a new method to determine the structure of integral membrane proteins by nuclear magnetic resonance. This new method also provides a unique view of membrane anchored proteins, some of which are potential targets for anti-viral therapies.