title: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
abstract: (13)C 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 (13)C 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 (13)C 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.
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Clark
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Zahm
authors.forename: Rustam
authors.initials: R
authors.lastname: Ali
authors.forename: Maciej
authors.initials: M
authors.lastname: Kukula
authors.forename: Liangqiao
authors.initials: L
authors.lastname: Bian
authors.forename: Steven M
authors.initials: SM
authors.lastname: Patrie
authors.forename: Kevin H
authors.initials: KH
authors.lastname: Gardner
authors.forename: Michael K
authors.initials: MK
authors.lastname: Rosen
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Rosenbaum
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry* / Q000378:metabolism; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000199:Actins; D002247:Carbon Isotopes; D011994:Recombinant Proteins; D007532:Isoleucine