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 structures. The proposed Center brings together 11 Investigators at five US institutions to focus cooperatively on the overarching aim of determining integral MP structures of high biomedical impact.
The Specific Aims balance multiple priorities.
Aims 1 -3 are extensive, seeking to obtain structures by providing many targets from (1) E. coli, (2) extremophiles, and (3) human. The broad target base is triaged by dynamic bioinformatics screening to direct focus on the most tractable set by the end of year 1.
Aims 4 and 5 are intensive, targeting families of highest biomedical relevance and impact for which structures have generally not yet been obtained;
Aim 4 concerns specific prokaryotic MPs;
Aim 5 involves the most challenging eukaryotic MPs, including human therapeutic targets and components of the nuclear pore complex.
Aim 6 leverages MP structures by comparative modeling developed specifically for MPs. Ten core capabilities implement the methods that support the aims and cover every aspect of structure determination, including target selection, cloning, expression, purification, crystallization, structure determination by X-ray crystallography, NMR spectroscopy or electron microscopy, and modeling. The cores provide multi-point entry to High-Throughput-Enabled Structural Biology Partnerships. Expression cores cover prokaryotic and eukaryotic (including HEKs) in vivo systems, one using green fluorescent protein detection of expression, and an E. coli based cell-free in vitro system optimized for MP expression. The protein purification core, aided by several characterization methods, provides pure homogeneous and stable proteins free of excess detergent. The electron microscopy core provides further characterization and 2D crystallization. Structure determination methods include X-ray diffraction and NMR spectroscopy, where cell-free expression has been harnessed to a combinatorial labeling strategy for rapid determination of backbone structures. The X-ray crystallography core provides robotic crystal trials and diffraction at the Advanced Light Source beam line 8.3.1, one of the world's most productive protein crystallography facilities. Overall, the combined expertise of principal investigators provides a unique environment to achieve the proposed aims.

Public Health Relevance

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 structures. The proposed Center brings together 11 Investigators at five US institutions to focus cooperatively on the overarching aim of determining integral MP structures of high biomedical impact.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
3U54GM094625-04S1
Application #
8718077
Study Section
Special Emphasis Panel (ZGM1-CBB-3 (MP))
Program Officer
Preusch, Peter C
Project Start
2010-09-30
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$159,277
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Holton, James M; Classen, Scott; Frankel, Kenneth A et al. (2014) The R-factor gap in macromolecular crystallography: an untapped potential for insights on accurate structures. FEBS J 281:4046-60
Pieper, Ursula; Webb, Benjamin M; Dong, Guang Qiang et al. (2014) ModBase, a database of annotated comparative protein structure models and associated resources. Nucleic Acids Res 42:D336-46
Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal et al. (2014) Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels. J Mol Biol 426:467-83
Miercke, Larry J W; Robbins, Rebecca A; Stroud, Robert M (2014) Tetra detector analysis of membrane proteins. Curr Protoc Protein Sci 77:29.10.1-29.10.30
Gong, Zhen; Kessans, Sarah A; Song, Lusheng et al. (2014) Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41. Protein Sci 23:1607-18
Monk, Brian C; Tomasiak, Thomas M; Keniya, Mikhail V et al. (2014) Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer. Proc Natl Acad Sci U S A 111:3865-70
Lang, P Therese; Holton, James M; Fraser, James S et al. (2014) Protein structural ensembles are revealed by redefining X-ray electron density noise. Proc Natl Acad Sci U S A 111:237-42
Weierstall, Uwe; James, Daniel; Wang, Chong et al. (2014) Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nat Commun 5:3309
Webb, Benjamin; Sali, Andrej (2014) Protein structure modeling with MODELLER. Methods Mol Biol 1137:1-15
Li, Dianfan; Lyons, Joseph A; Pye, Valerie E et al. (2013) Crystal structure of the integral membrane diacylglycerol kinase. Nature 497:521-4

Showing the most recent 10 out of 28 publications