DESCRIPTION (provided by applicant): The long-term objective is to engineer proteins 'designed to order'. The specific aim is to stabilize G protein-coupled receptors (GPCRs), which are membrane proteins. GPCRs transmit most cellular responses across cell membranes through a vast array of extracellular stimuli, which include small molecules, light, proteins, peptides, hormones, and ions. GPCRs are involved in almost every physiological process. Therefore, irregular control can lead to pathological conditions. As a result, GPCRs are major drug targets. Crystal structure determination is necessary to investigate the molecular details of activation/deactivation. However, there are few structures available because GPCRs are difficult to crystallize. For instance, they suffer stability problems due to flexibility. Hence, a robust an efficient protein engineering system is needed to optimize these proteins for structural determination. Current or traditional protein display technologies, which include phage, gram-negative bacteria, and eukaryotes, suffer from protein folding and viability issues. For example, GPCRs require detergents to solubilize and refold the proteins. However, traditional methods may not be compatible with these conditions. The initial GPCR target will be human parathyroid hormone receptor 1, which is vital in regulating calcium and phosphate levels in the blood. Bacillus subtilis spores will be developed as a protein display platform for direct or laboratory evolution. Spores tolerate many physical/chemical extremes. Hence, they remain viable during harsh screening conditions, which include detergents. Furthermore, protein folding issues that are associated with traditional protein display are avoided. Hence, spore display will be able to access a region of protein space that was previously unavailable. This research will aid in overcoming stability issues require for structural determination and facilitate the understanding of the structure/function relationships. In addition, the groundwork will be established for spore display as a general tool for protein engineering. Public HEALTH RELEVANCE: G protein-coupled receptors (GPCRs) transmit most cellular responses across cell membranes through a vast array of extracellular stimuli, and they are involved in almost every physiological process. Therefore, irregular control leads to pathological conditions. GPCRs will be evolved for enhanced stability in order to facilitate the understanding of activation and deactivation on the molecular level.
|Effective start/end date||5/1/12 → 4/30/16|
- National Institutes of Health
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