Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote <Emphasis Type="Italic">Pichia pastoris</Emphasis> |
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| Authors: | Lindsay Clark Jacob A Zahm Rustam Ali Maciej Kukula Liangqiao Bian Steven M Patrie Kevin H Gardner Michael K Rosen Daniel M Rosenbaum |
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| Institution: | 1.Department of Biophysics,University of Texas Southwestern Medical Center,Dallas,USA;2.Howard Hughes Medical Institute,University of Texas Southwestern Medical Center,Dallas,USA;3.Department of Pathology,University of Texas Southwestern Medical Center,Dallas,USA;4.Shimadzu Center for Advanced Analytical Chemistry,University of Texas at Arlington,Arlington,USA;5.Structural Biology Initiative,CUNY Advanced Science Research Center,New York,USA |
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| Abstract: | 13C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific 13C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient 13C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets. |
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