Mediating Membrane Protein Crystal Contacts by Stabilization Reagents

描述(申请人提供)：与可溶性蛋白质相比，获得适合X射线分析的衍射性良好的膜蛋白晶体更具挑战性。膜蛋白晶体的特征是小的亲水性蛋白质-蛋白质相互作用，这是形成3D晶格的关键。扩展或修饰膜蛋白的极性表面的方法对于晶体生长是有效的，但仍然是重大的挑战。我们建议的总体目标是开发一种创新的方法，使用智能设计的两亲性或脂类，稳定膜蛋白质所需的基本成分，以调节蛋白质表面的有序相互作用，从而增加结晶倾向和改善晶体衍射。该方法是现有蛋白质工程技术的正交性和互补性，既适用于洗涤剂胶束结晶，也适用于基于脂质双层的结晶方案。为了实现我们的目标，我们将为创造新型两亲性化合物开发新的设计原则。我们将使用生化分析和各种生物物理技术来研究两亲分子与膜蛋白之间的热力学相互作用和结合，因为这些性质决定了蛋白质的稳定性和功能。将进行结晶实验，以确定介导有序膜蛋白晶体接触的分子，并揭示两亲性蛋白质相互作用的分子细节。通过这项工作，结构新颖的稳定剂将被开发出来，以克服结晶瓶颈，而目前可用的洗涤剂、脂类或其他在过去20年中已经测试过的新型两亲性物质无法完全解决这一瓶颈。我们的目标是确定一套强大的试剂集，可以普遍适用于不同膜蛋白家族的结构溶液，而不是局限于单一蛋白质或单一类别的试剂。通过提高以前解决的结构的分辨率和促进新的膜蛋白的结构确定，我们的研究将对生物学产生直接的影响。

DESCRIPTION (provided by applicant): Compared to soluble proteins, it is much more challenging to obtain well diffracting membrane protein crystals suitable for X-ray analysis. Membrane protein crystals are characterized by small hydrophilic protein- protein interactions that are crucial for formation of a 3D crystal lattice. Approaches to expand or modify the polar surface of membrane proteins are effective for crystal growth but still represent significant challenges. The overarching goal of our proposal is to develop an innovative approach of using intelligently designed amphiphiles or lipids, the essential component required to stabilize membrane proteins, to mediate ordered protein surface interactions so as to increase the crystallization propensity and improve crystal diffraction. This approach is orthogonal and complementary to available protein engineering techniques and applicable to both detergent micelle and lipid bilayer based crystallization protocols. To achieve our goal, we will develop new design principles for the creation of novel amphiphiles. We will use biochemical assays and various biophysical techniques to study the thermodynamic interaction and binding between the amphiphiles and membrane proteins, as these properties govern protein stability and function. Crystallization experiments will be performed to identify molecules that mediate ordered membrane protein crystal contacts and reveal molecular details of the amphiphile-protein interaction. Through this work, structurally novel stabilization reagents will be developed to overcome the crystallization bottleneck that cannot be fully addressed by currently available detergents, lipids or other novel amphiphiles that have been tested in the last two decades. We aim to identify a robust set of reagents that can be generally applicable to the structural solution of different families of membrane proteins, not ones limited to a single protein or a single class. By improving the resolution of previously solved structures and facilitating the structural determination of new membrane proteins, our study will have a direct impact on biology.