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

描述（由申请人提供）：通过晶体学和核磁共振（NMR）确定的蛋白质高分辨率结构模型数量激增。然而，当已知结构的数量接近50，000时，其中只有几百个是完整的膜蛋白。在不久的将来，结构生物学的设想是在确定足够数量的独特蛋白质结构方面取得重大进展，以允许仅基于序列预测任何结构。不幸的是，这一极具挑战性的目标变得更加困难的重要障碍所提出的完整的膜蛋白，也许令人惊讶的是，膜锚定蛋白。该提案旨在摆脱这两类重要蛋白质的限制，并为通过溶液NMR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。我们的方法是不寻常的，而且仍在出现。这是基于我们早期的工作，使用反胶束封装，以克服缓慢翻滚的问题所提出的大可溶性蛋白质。在该方法中，感兴趣的蛋白质被封装在反胶束颗粒的保护性水性核内，并且整个组装体溶解在低粘度流体如液体乙烷中。在低粘度流体中，反胶束颗粒翻滚得比溶解在本体水中的蛋白质快。这提供了一个显着的改善NMR弛豫性能的现代三重共振实验的效率。该方法允许高性能的NMR光谱上获得的可溶性蛋白质大至100 kDa，而没有氘代或TROSY效应的好处。在这里，我们建议采用这种方法来研究积分和脂质锚定的外周膜蛋白。将使用反胶束法对Kcsa钾通道的跨膜段进行结构表征。这将为研究多肽链形成通道的动力学提供基础，这些动力学被认为对离子选择性很重要。与Kcsa的研究将设置一个测试的一般性的方法，以结构表征的完整的膜蛋白的阶段。与此同时，我们将采用反胶束包封研究共价连接的脂质在蛋白质锚定到膜的作用。我们将研究肉豆蔻酰化的HIV-1基质蛋白在其与磷酸肌醇PIP 2结合时的结构-功能关系。这种相互作用已被认为是药物干预的有前途的目标。经典的肉豆蔻基开关蛋白recoverin将用于进一步定义该方法。类似的研究，提出了棕榈酰化的蛋白质BET 3，膜靶向复合物的一个组成部分，和UL 11，单纯疱疹病毒1型的被膜蛋白。通常嵌入在膜中的蛋白质和配体之间的相互作用也将被研究。这些研究应建立反胶束增溶方法作为整体和脂质锚定膜蛋白的结构和动力学研究的一般方法。公共卫生相关性：虽然整合膜蛋白对人类生物学中的许多基本过程至关重要，并且代表了医学中药物干预的大多数靶点，但在结构细节上表征它们仍然非常困难。在这里，我们将开发一种新的方法来确定膜蛋白的结构，通过核磁共振。这种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.