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Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra

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Abstract

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 (13C/15N) isotopically labeled in this system and functionally reconstituted into phospholipids, giving excellent resolution of solid-state magic angle spinning NMR spectra.

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Acknowledgments

We thank Frederick Öberg and Kristina Hedfalk (Göteborg University, Sweden) for the generous gift of hAQP1 expression vector. The research was supported by the University of Guelph (start-up funds to V.L. and L.S.B.), the Natural Sciences and Engineering Research Council of Canada (discovery grants to L.S.B. and to V.L.), Canada Foundation for Innovation, and the Ontario Ministry of Research and Innovation. V.L. holds Canada Research Chair in biophysics, and S.E. is a recipient of the Ontario Trillium scholarship. We thank Drs. Armen Charchoglyan and Dyanne Brewer for MALDI-MS data collection. We thank Dr. Miguel Lugo for his help with stopped-flow measurements and Dr. Shenlin Wang for the help with NMR data processing. We thank Cambridge Isotope Laboratories for the generous gift of isotopically labeled methanol.

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  1. Departments of Physics, and Biophysics Interdepartmental Group, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada

    Sanaz Emami, Ying Fan, Rachel Munro, Vladimir Ladizhansky & Leonid S. Brown

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  1. Sanaz Emami
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  2. Ying Fan
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  3. Rachel Munro
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Correspondence to Leonid S. Brown.

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Emami, S., Fan, Y., Munro, R. et al. Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra. J Biomol NMR 55, 147–155 (2013). https://doi.org/10.1007/s10858-013-9710-5

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  • DOI: https://doi.org/10.1007/s10858-013-9710-5

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