Engineering membrane proteins for biophysical studies

描述(申请人提供)：G蛋白偶联受体(GPCRs)是生物和临床上最重要的一类蛋白质之一，占脊椎动物基因组的1%以上，占所有药物靶标的50%以上。有趣的是，这些受体的激活配体范围从完整的蛋白质到光子，但我们无法详细说明GPCRs的结构-功能关系，因为生成这些七个跨膜受体的高分辨率晶体结构非常困难。组合诱变策略将被用来直接解决生产适合于结晶学研究的GPCRs的两个最大限制，即受体晶体的低表达/稳定性和不充分的衍射质量。首先，容易出错的聚合酶链式反应方法将被用来产生一个受体文库，该文库可以转化到细菌中，并使用选择标记，如荧光蛋白融合或必要的酶来筛选表达水平。然后，这个较小的文库可以通过在洗涤剂溶液中增溶并针对固定的配体进行筛选，从而测试膜外受体的稳定性以及配体结合的能力。其次，核糖体展示将被用来选择与GPCRs上特定表位结合的新的Anyrin-Repeat蛋白。这些部分可以用作GPCR结晶的佐剂，因为很容易结晶的Ankyrin-Repeat蛋白可以诱导形成共晶格，从而极大地改善了衍射质量。这项工作将产生广泛的影响，从受体生物学和信号转导的基础研究到基于结构的药物设计的临床应用。

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) are one of the most biologically and clinically important class of proteins, representing greater than 1% of the vertebrate genomes and greater than 50% of all pharmaceutical targets. Intriguingly, the activating ligands for these receptors range from full proteins to photons, yet we are unable to detail the structure-function relationships for GPCRs because of the great difficulty in generating high resolution crystal structures of these seven-transmembrane receptors. Combinatorial mutagenesis strategies will be employed to directly address two of the biggest limitations in producing GPCRs suitable for crystallographic studies, low expression/stability and inadequate diffraction quality of receptor crystals. First, error-prone polymerase chain reaction methods will be used to generate a receptor library that can be transformed into bacteria and screened for expression level using a selection marker such as a fluorescent protein fusion or an essential enzyme. This smaller library can then by solubilized in detergent solution and screened against immobilized ligand, thereby testing for receptor stability out of the membrane as well as competency in ligand binding. Second, ribosome display will be used to select novel ankyrin-repeat proteins that bind to specific epitopes on GPCRs. Such moieties may be used as adjuvants for GPCR crystallization, since ankyrin-repeat proteins, which readily crystallize, may induce a co-crystal lattice formation that has a greatly improved diffraction quality. This work will have a wide-reaching impact, ranging from fundamental studies on receptor biology and signal transduction to clinical applications in structure-based drug design.