Engineering membrane proteins for biophysical studies

描述（由申请人提供）：G蛋白偶联受体（GPCR）是生物学和临床上最重要的一类蛋白质，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，这些受体的活化配体范围从完整的蛋白质到光子，但我们无法详细说明GPCR的结构-功能关系，因为很难生成这些七跨膜受体的高分辨率晶体结构。将采用组合诱变策略来直接解决生产适合于晶体学研究的GPCR中的两个最大限制，即低表达/稳定性和受体晶体的衍射质量不足。首先，将使用易错聚合酶链反应方法来产生受体文库，所述受体文库可以转化到细菌中并使用选择标记（例如荧光蛋白融合物或必需酶）筛选表达水平。然后可以将该较小的文库溶解在去污剂溶液中并针对固定的配体进行筛选，从而测试膜外的受体稳定性以及配体结合的能力。其次，核糖体展示将用于选择新的锚定重复序列蛋白，结合到GPCR上的特定表位。这样的部分可以用作GPCR结晶的佐剂，因为容易结晶的锚定重复序列蛋白可以诱导具有大大改善的衍射质量的共晶格形成。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.