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）确定的高分辨率蛋白质结构模型的数量激增。然而，当已知结构的数量接近5万个时，其中只有几百个是完整的膜蛋白。在不久的将来，结构生物学预计将在确定足够数量的独特蛋白质结构方面取得重大进展，从而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由于整体膜蛋白和膜锚定蛋白（也许令人惊讶）所呈现的重要屏障而变得更加困难。该建议旨在摆脱这两类重要蛋白质的限制，并为通过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，而且仍在发展中。这是基于我们早期的工作，使用反胶束封装来克服大的可溶性蛋白质所带来的缓慢翻滚问题。在这种方法中，感兴趣的蛋白质被封装在反胶束颗粒的保护性水性核心中，整个组件溶解在低粘度流体中，如液态乙烷。在低粘度流体中，反向胶束颗粒比溶解在散装水中的蛋白质翻滚得快。这为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。该方法可以获得高达100 kDa的可溶性蛋白的高性能核磁共振光谱，而不需要氘化或TROSY效应。在这里，我们建议将这种方法应用于整体和脂质锚定外周膜蛋白的研究。反胶束法将用于表征Kcsa钾通道跨膜段的结构。这将为多肽链形成通道的动力学研究提供基础，这被认为是离子选择性的重要研究。Kcsa的研究将为测试整体膜蛋白结构表征方法的普遍性奠定基础。同时，我们将采用反胶束封装来研究共价附着的脂质在蛋白质锚定到膜上的作用。我们将研究豆蔻酰基化的HIV-1基质蛋白与磷酸肌苷PIP2结合的结构-功能关系。这种相互作用已被提出为药物干预的有希望的目标。经典的肉豆蔻酰基开关蛋白恢复将用于进一步定义该方法。类似的研究也被提出用于棕榈酰化蛋白BET3（一种膜靶向复合物的成分）和UL11（一种单纯疱疹病毒的被膜蛋白）。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。这些研究应该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：尽管整体膜蛋白对人类生物学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目标，但在结构细节上表征它们仍然非常困难。本文将发展一种利用核磁共振测定整体膜蛋白结构的新方法。这种新方法也为膜锚定蛋白提供了一个独特的视角，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.