We demonstrate a novel sparse 13C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically 13C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.
[NMR paper] Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Related Articles Erratum to: Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
J Biomol NMR. 2016 Feb 19;
Authors: Clark L, Zahm JA, Ali R, Kukula M, Bian L, Patrie SM, Gardner KH, Rosen MK, Rosenbaum DM
PMID: 26894386
[NMR paper] Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR.
Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR.
Related Articles Isotope Labeling of Eukaryotic Membrane Proteins in Yeast for Solid-State NMR.
Methods Enzymol. 2015;565:193-212
Authors: Fan Y, Emami S, Munro R, Ladizhansky V, Brown LS
Abstract
Solid-state NMR (ssNMR) is a rapidly developing technique for exploring structure and dynamics of membrane proteins, but its progress is hampered by its low sensitivity. Despite the latest technological advances, routine ssNMR experiments still require...
nmrlearner
Journal club
0
11-19-2015 05:22 PM
[NMR paper] Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
Related Articles Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.
J Biomol NMR. 2015 May 30;
Authors: Clark L, Zahm JA, Ali R, Kukula M, Bian L, Patrie SM, Gardner KH, Rosen MK, Rosenbaum DM
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...
nmrlearner
Journal club
0
05-31-2015 01:57 PM
Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris
Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris
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...
nmrlearner
Journal club
0
05-29-2015 01:24 PM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Abstract Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives,...
nmrlearner
Proteins
0
01-22-2011 03:46 AM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
J Biomol NMR. 2011 Jan 19;
Authors: Fan Y, Shi L, Ladizhansky V, Brown LS
Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties...
Sparse 13 C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins
Linear Mode
