This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Integral membrane proteins account for ~30% of a proteome and play critical roles in metabolic, regulatory and intercellular processes. Human MPs are the targets for ~40% of all therapeutic drugs, but the number of MP structures is less than 0.5% of the number of soluble protein 3D structures. In our lab and as members of the Center for Structure of Membrane Proteins (CSMP) and Membrane Protein Expression Center (MPEC), we aim to determine integral MP structures of high biomedical impact. Our centers strive to obtain structures by providing many targets from E. coli, extremophiles, yeast and humans. Our expression capabilities include prokaryotic and eukaryotic (including HEKs) in vivo systems, and an E. coli based cell-free in vitro system optimized for MP expression. The protein purification core lab includes characterization methods and provides pure homogeneous and stable proteins free of excess detergent. Structure determination by include X-ray diffraction is aided by robotic crystal trials and diffraction at the Advanced Light Source beamline 8.3.1, one of the world's most productive protein crystallography facilities. Mass Spectroscopy analysis will be useful in confirming each protein mass and homogeneity, since some membrane protein have cleaved N-termini or are subject to post-translational modification.
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