DETERMINANTS OF GPCR EXPRESSION IN E COLI AND YEAST

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 即使有足够的表达水平，膜蛋白的研究也受到提取和有时折叠蛋白质的困难的限制。因此，我们将建立一个策略来选择最适合纯化、增溶和稳定天然结构的洗涤剂和脂类。这将包括研究表面活性剂体系本身的性质，特别是相行为，以及它们在膜蛋白存在时的行为。除了指导重新折叠战略外，这些信息本身也将是有用的。我们的结果将增加对膜蛋白插入、折叠以及与洗涤剂和脂类相互作用机制的了解，并将促进大量活性完整膜蛋白的生产。 本工作的目标是在酿酒酵母中设计一个异源的GPCR表达系统，以获得高水平的纯化的、具有功能的G蛋白偶联受体，用于生物物理特性和高分辨结构的确定。为了实现这一目标，我们试图通过仔细研究受体的运输和折叠，以及在表达过程中可能触发的细胞内应激反应，来更好地表征酿酒酵母作为宿主系统的特征。通过确定在酵母中高水平表达GPCRs的条件，该系统将成为促进哺乳动物GPCRs过度表达和生物物理特性的更理想的宿主。 该项目的目标如下： -使用生物物理方法表征人的腺苷A2a受体 -工程酿酒酵母表达毫克量的活性哺乳动物GPCRs 这个子项目还涵盖了最初包含在SPID 0021中但在赠款的较早时候包含在SPID 0020中的主题。这项研究包括建立一种策略来选择最适合于膜蛋白天然结构的纯化、增溶和稳定的洗涤剂和脂类。还将研究蛋白质结晶中使用的含有添加剂的表面活性剂溶液的相行为，以指导更可靠地结晶膜蛋白质的努力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Even with adequate expression levels, the study of membrane proteins is limited by difficulties in extracting and sometimes refolding the proteins. We will therefore establish a strategy for choosing detergents and lipids optimal for purification, solubilization, and stabilization of native structure. This will include studies of the properties, particularly phase behavior, of the surfactant systems themselves, as well as of their behavior in the presence of membrane proteins. In addition to guiding refolding strategies, this information will be useful in its own right. Our results will provide increased knowledge of the mechanism of membrane protein insertion, folding, and interactions with detergents and lipids, and will facilitate production of significant quantities of active integral membrane proteins. The goal of this work is to engineer a heterologous GPCR expression system in the yeast S. cerevisiae in order to obtain high levels of purified, functional G-protein coupled receptors for biophysical characterization and high-resolution structure determination. To achieve this goal, we seek to better characterize S. cerevisiae as a host system by carefully examining receptor trafficking and folding, as well as intracellular stress responses that may be triggered during expression. By identifying the conditions that allow for high levels of functional expression of GPCRs in yeast, this system will serve as a more desirable host to accelerate mammalian GPCR over-expression and biophysical characterization. The goals of this project are listed below: -Characterize the human adenosine A2a receptor using biophysical methods -Engineer S. cerevisiae to express milligram amounts of active mammalian GPCRs This subproject also covers topics originally included in SPID 0021 but incorporated in SPID 0020 earlier in the grant. This research involves establishing a strategy for choosing detergents and lipids optimal for purification, solubilization, and stabilization of native structure in membrane proteins. The phase behavior of surfactant solutions containing additives used in protein crystallization will also be investigated in order to guide efforts to crystallize membrane proteins more reliably.