Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra.
One of the biggest challenges in solid-state NMR studies of membrane proteins is to obtain a homogeneous natively folded sample giving high spectral resolution sufficient for structural studies. Eukaryotic membrane proteins are especially difficult and expensive targets in this respect. Methylotrophic yeast Pichia pastoris is a reliable producer of eukaryotic membrane proteins for crystallography and a promising economical source of isotopically labeled proteins for NMR. We show that eukaryotic membrane protein human aquaporin 1 can be doubly ((13)C/(15)N) isotopically labeled in this system and functionally reconstituted into phospholipids, giving excellent resolution of solid-state magic angle spinning NMR spectra.
Departments of Physics, and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada.
Sanaz
S
Emami
Ying
Y
Fan
Rachel
R
Munro
Vladimir
V
Ladizhansky
Leonid S
LS
Brown
D051398:Aquaporin 1 / Q000737:chemistry* / Q000235:genetics; D002247:Carbon Isotopes; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008081:Liposomes; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared
C496020:AQP1 protein, human; D002247:Carbon Isotopes; D008081:Liposomes; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D051398:Aquaporin 1