This project aims to develop protocols that will lead to the establishment of a robust high-throughput pipelinefor the atomic-level structural characterization of membrane protein microcrystals grown in membrane-likeenvironment of lipidic cubic phase (LCP) using serial femtosecond nanocrystallography (SFX) at free electronX-ray laser sources (XFELs). With the use of SFX, we will obviate the need for obtaining large crystals,effectively eliminate radiation damage issues through 'diffraction before destruction' (i.e., diffraction data arecollected prior to onset of any damage), simplify handling, as harvesting and freezing are not required, andsignificantly reduce the time from obtaining initial crystal hits to collecting full data sets. Our long term goal isthe integration of this technology into our structural determination pipeline enabling the determination of a largenumber of three-dimensional structures of G protein-coupled receptors (GPCRs)-ligand complexes addressingquestions on ligand selectivity and efficacy using structure-based drug discovery (SBDD) approaches. Ourgoal will be achieved through the following specific aims.
Aim 1 : Develop protocols for the production ofsamples of GPCR-ligand complexes and for the generation, and characterization of large number ofmicrocrystals that can be used for SFX studies.
Aim 2 : Develop protocols for SFX data collection, processingand structure solution of GPCR-ligand complexes.
Aim 3 : Integrate protocols developed in Aims 1 and 2 intothe GPCR Structure Determination Pipeline and optimize and validate the modified pipeline by determining thestructure of novel GPCRs including a number of receptor-ligand complexes. GPCRs constitute the largestfamily of membrane proteins in the human genome with approximately 800 members and are responsible fortransmitting variety of extracellular signals inside the cell, thereby controlling all major physiological responses,including vision, olfactory, immune defense, reproduction, digestion, mental behavior and others; severalGPCRs are exploited as co-receptors for entry by HIV and other pathogens. GPCR signaling through multipleeffector pathways has profound therapeutic implications, which underscores the need to understand thereceptor both biochemically and structurally in the proper context. GPCRs are the target of ~40% of currentlymarketed drugs. However, detailed understanding of their mechanism of action and ligand selectivity is limitedby a lack of structural information. The structure determination of GPCRs is hampered by the difficulty ofpreparing large amounts of homogenous and stable samples and growing sufficiently large crystals for high-resolution structure determination even when using state-of-the art microfocus beamlines at synchrotronsources.
The proposed studies will develop protocols that will enable high-throughput determination of high-resolution structures of G protein-coupled receptors and other membrane proteins; which are an important class of drug targets for diverse pathophysiological conditions in vision; olfactory; immune defense; reproduction; digestion; mental behavior; and others including cancers.
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