Mediating Membrane Protein Crystal Contacts by Stabilization Reagents

通过稳定试剂介导膜蛋白晶体接触

• 批准号: 8331612
• 负责人: Qinghai Zhang
• 金额: $ 36.01万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8331612
• 关键词: Accounting; Address; Antibodies; Area; Bile Acids; Binding; Biochemical; Biological; Biological Assay; Biology; Cell membrane; Chemicals; Chimeric Proteins; Cholesterol; Collaborations; Complex; Crystallization; Crystallography; Detergents; Development; Drug Delivery Systems; Drug Design; Entropy; Epitopes; Family; Fluorescence; Genes; Goals; Growth; Human; Hydrogen Bonding; Hydrophobic Surfaces; Imagery; Integral Membrane Protein; Ion Channel; Label; Laboratories; Lipid Bilayers; Lipids; Literature; Measures; Mediating; Membrane Proteins; Methodology; Methods; Micelles; Modification; Molecular; Outcome; Process; Property; Protein Engineering; Proteins; Protocols documentation; Reagent; Reporting; Resolution; Roentgen Rays; Side; Solutions; Specificity; Steroids; Sterols; Structure; Surface; Techniques; Testing; Thermodynamics; Work; base; chemical group; design; flexibility; improved; innovation; molecular recognition; novel; novel strategies; protein protein interaction; protein structure; research study

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.

描述（申请人提供）：与可溶性蛋白质相比，获得适合X射线分析的良好衍射的膜蛋白质晶体更具挑战性。膜蛋白晶体的特点是小的亲水性蛋白质-蛋白质相互作用，这对于 3D 晶格的形成至关重要。扩展或修饰膜蛋白极性表面的方法对于晶体生长来说是有效的，但仍然存在重大挑战。我们提案的总体目标是开发一种创新方法，使用智能设计的两亲物或脂质（稳定膜蛋白所需的基本成分）来介导有序的蛋白质表面相互作用，从而增加结晶倾向并改善晶体衍射。该方法与现有的蛋白质工程技术是正交和补充的，并且适用于基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来创造新型两亲物。我们将使用生化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，因为这些特性控制着蛋白质的稳定性和功能。将进行结晶实验来鉴定介导有序膜蛋白晶体接触的分子，并揭示两亲物-蛋白质相互作用的分子细节。通过这项工作，将开发出结构新颖的稳定试剂，以克服目前可用的去污剂、脂质或过去二十年来经过测试的其他新型两亲物无法完全解决的结晶瓶颈。我们的目标是确定一组强大的试剂，这些试剂可以普遍适用于不同膜蛋白家族的结构解决方案，而不仅限于单一蛋白质或单一类别。通过提高先前解决的结构的分辨率并促进新膜蛋白的结构测定，我们的研究将对生物学产生直接影响。