DETERMINANTS OF GPCR EXPRESSION IN E COLI AND YEAST

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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.