The Transmembrane Protein Center (TMPC) proposes to function as collaborative project within the PShBiology Network. The TMPC research team has been assembled to provide diverse skills and experience in all constituent tasks of structural genomics. Our collective experience suggests that application of many techniques will be needed to expand the chances for success with the broad range of membrane proteins expected from participation in the PShBiology Network. This project is wellpoised to provide the expertise and leadership needed to successfully advance the goals of the PSLBiology Network, including outreach to new participants in this large-scale endeavor. The assembled team includes scientists with experience in the expression, production, and characterization of membrane proteins, proven ability to solve structures of complicated membrane proteins, and a strong track record of studying the interactions of membrane proteins with their protein partners and biological ligands. The team has strong grounding in the use of bioinformatics and bioanalytical techniques. Every participant brings unique experience with the function of membrane proteins, including assay development, preparation methods, or studies with either purified proteins and living organisms. This emphasis on merging the production of membrane proteins for structure determination with the power of functional characterizations is the central philosophy of our Center. The TMPC has a primary goal of determining a founding structure for each ofthe unique target classes elaborated in this proposal. Our second goal is to establish an efficient membrane protein production pipeline so that increases in throughput may be progressively obtained. The TMPC expects to improve its ability to create protein variants, to refine screening based on functional and biophysical characterizations, and to improve sorting between multiple expression pathways to find the best way produce a protein of interest. Emphasis on small-scale purification screening, and further integration of automation into our efforts will increase efficiency. Expanded options for crystallization screening, access to synchrotrons, and improvements in software used to solve structures will also contribute to the increased throughput necessary to achieve PSi:Biology goals.

Public Health Relevance

This project provides basic research on integral membrane proteins, which are found in all living organisms. This work contributes to the appreciation of biological processes and gives unique insight into how these proteins contribute to human health and wellbeing. Methods developed in this research may also lead to better treatments for diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54GM094584-05
Application #
8699208
Study Section
Special Emphasis Panel (ZGM1-CBB-3)
Project Start
Project End
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
5
Fiscal Year
2014
Total Cost
$836,619
Indirect Cost
$273,240
Name
University of Wisconsin Madison
Department
Type
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Aoki, Scott T; Porter, Douglas F; Prasad, Aman et al. (2018) An RNA-Binding Multimer Specifies Nematode Sperm Fate. Cell Rep 23:3769-3775
Minkoff, Benjamin B; Makino, Shin-Ichi; Haruta, Miyoshi et al. (2017) A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA. J Biol Chem 292:5932-5942
Alderson, Thomas Reid; Kim, Jin Hae; Markley, John Lute (2016) Dynamical Structures of Hsp70 and Hsp70-Hsp40 Complexes. Structure 24:1014-30
Li, Bo; Makino, Shin-Ichi; Beebe, Emily T et al. (2016) Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein. Protein Expr Purif 126:33-41
Lee, Kanghyun; Sharma, Ruchika; Shrestha, Om Kumar et al. (2016) Dual interaction of the Hsp70 J-protein cochaperone Zuotin with the 40S and 60S ribosomal subunits. Nat Struct Mol Biol 23:1003-1010
Stefely, Jonathan A; Licitra, Floriana; Laredj, Leila et al. (2016) Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity. Mol Cell 63:608-620
Aoki, Scott T; Kershner, Aaron M; Bingman, Craig A et al. (2016) PGL germ granule assembly protein is a base-specific, single-stranded RNase. Proc Natl Acad Sci U S A 113:1279-84
Aceti, David J; Bingman, Craig A; Wrobel, Russell L et al. (2015) Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies. J Struct Funct Genomics 16:67-80
Bai, Yonghong; McCoy, Jason G; Levin, Elena J et al. (2015) X-ray structure of a mammalian stearoyl-CoA desaturase. Nature 524:252-6
Beebe, Emily T; Makino, Shin-Ichi; Markley, John L et al. (2014) Automated cell-free protein production methods for structural studies. Methods Mol Biol 1140:117-35

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