caa a22 4500 445325070 CHVBK 20180317142717.0 cr unu---uuuuu 170323e20110201xx s 000 0 eng 10.1007/s10858-011-9473-9 doi (NATIONALLICENCE)springer-10.1007/s10858-011-9473-9 Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment [Elektronische Daten] [Ying Fan, Lichi Shi, Vladimir Ladizhansky, Leonid Brown] 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, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5mg of purified uniformly 13C,15N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution 13C and 15N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods. Springer Science+Business Media B.V., 2011 Solid-state NMR nationallicence Magic angle spinning nationallicence Uniformly 13C, 15N labeled proteins nationallicence Eukaryotic membrane proteins nationallicence Pichia pastoris nationallicence 7TM : Seven-transmembrane nationallicence ASR : Anabaena sensory rhodopsin nationallicence BR : Bacteriorhodopsin nationallicence BMD : Buffered minimal dextrose nationallicence BMM : Buffered minimal methanol nationallicence CHES : N-Cyclohexyl-2-aminoethanesulfonic acid nationallicence DMPC : 1,2-dimyristoyl-sn-glycero-3-phosphocholine nationallicence DMPA : 1,2-dimyristoyl-sn-glycero-3-phosphate nationallicence DTT : Dithiothreitol nationallicence E. coli : Escherichia coli nationallicence EDTA : Ethylenediaminetetraacetic acid nationallicence FTIR : Fourier-transform infrared nationallicence GPCRs : G-protein coupled receptors nationallicence LR : Leptosphaeria rhodopsin nationallicence MALDI TOF : Matrix-assisted laser desorption/ionization time-of-flight nationallicence MAS : Magic angle spinning nationallicence NR : Neurospora rhodopsin nationallicence P. pastoris : Pichia pastoris nationallicence PMSF : Phenylmethylsulfonyl fluoride nationallicence PR : Proteorhodopsin nationallicence SDS-PAGE : Sodium dodecyl sulfate polyacrylamide gel electrophoresis nationallicence SR-II : Sensory rhodopsin II nationallicence ssNMR : Solid-state NMR nationallicence YPD : Yeast peptone dextrose nationallicence Fan Ying Department of Physics, University of Guelph, 50 Stone Road East, N1G 2W1, Guelph, ON, Canada aut Shi Lichi Department of Physics, University of Guelph, 50 Stone Road East, N1G 2W1, Guelph, ON, Canada aut Ladizhansky Vladimir Department of Physics, University of Guelph, 50 Stone Road East, N1G 2W1, Guelph, ON, Canada aut Brown Leonid Department of Physics, University of Guelph, 50 Stone Road East, N1G 2W1, Guelph, ON, Canada aut Journal of Biomolecular NMR Springer Netherlands 49/2(2011-02-01), 151-161 0925-2738 49:2<151 2011 49 10858 https://doi.org/10.1007/s10858-011-9473-9 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9473-9 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Fan Ying Department of Physics, University of Guelph, 50 Stone Road East, N1G 2W1, Guelph, ON, Canada aut NATIONALLICENCE 700 1- Shi Lichi Department of Physics, University of Guelph, 50 Stone Road East, N1G 2W1, Guelph, ON, Canada aut NATIONALLICENCE 700 1- Ladizhansky Vladimir Department of Physics, University of Guelph, 50 Stone Road East, N1G 2W1, Guelph, ON, Canada aut NATIONALLICENCE 700 1- Brown Leonid Department of Physics, University of Guelph, 50 Stone Road East, N1G 2W1, Guelph, ON, Canada aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 49/2(2011-02-01), 151-161 0925-2738 49:2<151 2011 49 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer