title: Isotope labeling of eukaryotic membrane proteins in yeast for solid-state NMR.
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 several milligrams of isotopically labeled protein. While production of bacterial membrane proteins on this scale is usually feasible, obtaining such quantities of eukaryotic membrane proteins is often impossible or extremely costly. We have demonstrated that, by using isotopic labeling in yeast Pichia pastoris, one can inexpensively produce milligram quantities of doubly labeled functional samples, which yield multidimensional ssNMR spectra of high resolution suitable for detailed structural investigation. This was achieved by combining protocols of economical isotope labeling of soluble proteins previously used for solution NMR with protocols of expression of eukaryotic membrane proteins successfully employed for other methods. We review two cases of such isotope labeling, of fungal rhodopsin from Leptosphaeria maculans and human aquaporin-1.
authors.affiliation: Department of Physics, University of Guelph, Guelph, Ontario, Canada; Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Ying
authors.initials: Y
authors.lastname: Fan
authors.affiliation: Department of Physics, University of Guelph, Guelph, Ontario, Canada; Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Sanaz
authors.initials: S
authors.lastname: Emami
authors.affiliation: Department of Physics, University of Guelph, Guelph, Ontario, Canada; Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Rachel
authors.initials: R
authors.lastname: Munro
authors.affiliation: Department of Physics, University of Guelph, Guelph, Ontario, Canada; Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Ladizhansky
authors.affiliation: Department of Physics, University of Guelph, Guelph, Ontario, Canada; Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, Canada. Electronic address: lebrown@uoguelph.ca.
authors.forename: Leonid S
authors.initials: LS
authors.lastname: Brown
mesh_terms: D005656:Fungal Proteins / Q000737:chemistry*; D007553:Isotope Labeling; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010843:Pichia / Q000737:chemistry*
chemical_list: D005656:Fungal Proteins; D008565:Membrane Proteins
keywords: Eukaryotic membrane proteins; Human aquaporin-1; Isotope labeling; Pichia pastoris; Rhodopsin; Solid-state NMR; Yeast