The overall goals of this project are to develop and apply new methodology to obtain the atomic-resolution structure of membrane proteins by solid-state NMR (SSNMR). The new techniques include sample preparation, isotopic labeling strategies, high-field (750 MHz 1H frequency) magic-angle spinning NMR spectroscopy and multidimensional dipolar recoupling pulse sequences. Our preliminary data demonstrate that we can obtain high quality 2D and 3D SSNMR spectra of E. coli cytochrome bo3 ubiquinol oxidase, a large (~144 kDa) integral membrane protein complex. The enzyme was uniformly labeled with 13C and 15N, then purified and pelleted with endogenous membrane lipids. Excellent spectra can be obtained with approximately 50 nmol (approximately 8 mg) of purified protein (using 12 to 36 hours of measurement time for 2D experiments, or 24 to 96 hours for 3D experiments). The protein-lipid complex maintains the protein in an active, native conformation and provides a stabilizing environment for the protein. The sample shows no evidence of degradation after weeks of NMR data acquisition. Our data demonstrate that SSNMR has the immediate potential to interrogate selected parts of very large uniformly 13C,15N -labeled membrane proteins, and to provide complete 3D structures of smaller membrane proteins (approximatley 200 residues). To realize this potential is the goal of the proposed project. Improved methods for structure determination by SSNMR may relieve a serious bottleneck in the membrane protein structure determination pipeline.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM075937-04S1
Application #
7686473
Study Section
Special Emphasis Panel (ZGM1-PPBC-3 (MP))
Program Officer
Preusch, Peter C
Project Start
2005-09-23
Project End
2010-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
4
Fiscal Year
2008
Total Cost
$10,015
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Sperling, Lindsay J; Tang, Ming; Berthold, Deborah A et al. (2013) Solid-state NMR study of a 41 kDa membrane protein complex DsbA/DsbB. J Phys Chem B 117:6052-60
Tang, Ming; Nesbitt, Anna E; Sperling, Lindsay J et al. (2013) Structure of the disulfide bond generating membrane protein DsbB in the lipid bilayer. J Mol Biol 425:1670-82
Tang, Ming; Comellas, Gemma; Rienstra, Chad M (2013) Advanced solid-state NMR approaches for structure determination of membrane proteins and amyloid fibrils. Acc Chem Res 46:2080-8
Brothers, Michael C; Nesbitt, Anna E; Hallock, Michael J et al. (2012) VITAL NMR: using chemical shift derived secondary structure information for a limited set of amino acids to assess homology model accuracy. J Biomol NMR 52:41-56
Iwasaki, Toshio; Fukazawa, Risako; Miyajima-Nakano, Yoshiharu et al. (2012) Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin. J Am Chem Soc 134:19731-8
Zhou, Donghua H; Nieuwkoop, Andrew J; Berthold, Deborah A et al. (2012) Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy. J Biomol NMR 54:291-305
Morrissey, James H; Tajkhorshid, Emad; Sligar, Stephen G et al. (2012) Tissue factor/factor VIIa complex: role of the membrane surface. Thromb Res 129 Suppl 2:S8-10
Tang, Ming; Sperling, Lindsay J; Berthold, Deborah A et al. (2011) High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data. J Biomol NMR 51:227-33
Higman, Victoria A; Varga, Krisztina; Aslimovska, Lubica et al. (2011) The conformation of bacteriorhodopsin loops in purple membranes resolved by solid-state MAS NMR spectroscopy. Angew Chem Int Ed Engl 50:8432-5
Tang, Ming; Berthold, Deborah A; Rienstra, Chad M (2011) Solid-State NMR of a Large Membrane Protein by Paramagnetic Relaxation Enhancement. J Phys Chem Lett 2:1836-1841

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