Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins |
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Authors: | Richard C. Page Jacob D. Moore Hau B. Nguyen Mukesh Sharma Rose Chase Fei Philip Gao Charles K. Mobley Charles R. Sanders Liping Ma Frank D. Sönnichsen Sangwon Lee Stanley C. Howell Stanley J. Opella Timothy A. Cross |
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Affiliation: | (1) Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, USA;(2) National High Magnetic Field Laboratory, 1800 E. Paul Dirac Drive, Tallahassee, Florida 32310, USA;(3) Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306-4380, USA;(4) Department of Biochemistry and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232-8725, USA;(5) Department of Physiology & Biophysics, and the Cleveland Center for Structural Biology, Case Western Reserve University, Cleveland, Ohio 44106, USA;(6) Department of Chemistry and Biochemistry, University of California, San Diego, CA 92093-0307, USA |
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Abstract: | The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1–4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins. |
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Keywords: | HSQC Integral Membrane Proteins NMR Sample Preparation Structural Genomics TROSY |
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