Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.
We demonstrate a novel sparse (13)C 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 (13)C 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.
Beijing NMR Centre, Peking University, Beijing, China.|College of Chemistry and Molecular Engineering, Peking University, Beijing, China.|Beijing National Laboratory for Molecular Sciences, Beijing, China.
Jing
J
Liu
College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
Chang
C
Liu
The Scripps Research Institute, La Jolla, CA, 92037, USA.|Department of Physics, University of Guelph, Guelph, ON, Canada.
Ying
Y
Fan
Department of Physics, University of Guelph, Guelph, ON, Canada.|Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.
Rachel A
RA
Munro
Department of Physics, University of Guelph, Guelph, ON, Canada.|Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.
Vladimir
V
Ladizhansky
Department of Physics, University of Guelph, Guelph, ON, Canada.|Biophysics Interdepartmental Group, University of Guelph, Guelph, ON, Canada.
Leonid S
LS
Brown
Beijing NMR Centre, Peking University, Beijing, China. wangshenlin@pku.edu.cn.|College of Chemistry and Molecular Engineering, Peking University, Beijing, China. wangshenlin@pku.edu.cn.|Beijing National Laboratory for Molecular Sciences, Beijing, China. wangshenlin@pku.edu.cn.
Shenlin
0000-0002-0153-0578
S
Wang
D066241:Carbon-13 Magnetic Resonance Spectroscopy / Q000379:methods; D005057:Eukaryotic Cells; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011994:Recombinant Proteins; D015003:Yeasts / Q000235:genetics
D008565:Membrane Proteins; D011994:Recombinant Proteins
Eukaryotic seven-transmembrane proteins; Magic-angle-spinning solid-state NMR; P. pastoris expression system; Sparse 13C isotope labelling