Mediating Membrane Protein Crystal Contacts by Stabilization Reagents

描述（由申请人提供）：与可溶性蛋白相比，获得适合X射线分析的良好衍射的膜蛋白晶体更具挑战性。膜蛋白质晶体的特征在于小的亲水性蛋白质-蛋白质相互作用，这对于形成3D晶格至关重要。扩大或修饰膜蛋白极性表面的方法对于晶体生长是有效的，但仍然代表着重大挑战。我们的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物或脂质，所需的稳定膜蛋白的基本组成部分，介导有序的蛋白质表面相互作用，以增加结晶倾向，提高晶体衍射。这种方法是正交的和互补的可用的蛋白质工程技术和适用于洗涤剂胶束和脂质双层的结晶方案。为了实现我们的目标，我们将开发新的设计原则，以创造新的两亲物。我们将使用生物化学测定和各种生物物理技术来研究两亲物和膜蛋白之间的热力学相互作用和结合，因为这些特性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质晶体接触的分子，并揭示两亲物-蛋白质相互作用的分子细节。通过这项工作，将开发结构新颖的稳定化试剂，以克服结晶瓶颈，这些瓶颈不能完全解决目前可用的洗涤剂，脂质或其他在过去二十年中测试过的新型两亲物。我们的目标是确定一套强大的试剂，可以普遍适用于不同家族的膜蛋白的结构解决方案，而不是局限于一个单一的蛋白质或一类。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测定，我们的研究将对生物学产生直接影响。

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.