title: Exploiting hydrophobicity for efficient production of transmembrane helices for structure determination by NMR spectroscopy.
abstract: Despite the biological and pharmaceutical significance of membrane proteins, their tertiary structures constitute less than 3% of known structures. One of the major obstacles for initiating structural studies of membrane proteins by NMR spectroscopy is the generation of high amounts of isotope-labeled protein. In this work, we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations of the strategy using seven membrane protein variants that differ in their overall hydrophobicity and length and show a recovery for suitable variants of >70%. The developed production scheme is cost-efficient and easy to implement and has the potential to facilitate an increase in the number of structures of single-pass TMDs, which are difficult to solve by other means.
authors: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen , Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors: Katrine
authors: K
authors: Bugge
authors: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen , Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors: Helena
authors: H
authors: Steinocher
authors: The University of Queensland Diamantina Institute, The University of Queensland, Translational Research Institute, Brisbane, Queensland 4072, Australia.|The University of Queensland , Institute for Molecular Bioscience, Brisbane, Queensland 4072, Australia.
authors: Andrew J
authors: AJ
authors: Brooks
authors: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen , Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors: Kresten
authors: K
authors: Lindorff-Larsen
authors: Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen , Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors: Birthe B
authors: BB
authors: Kragelund
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010742:Phospholipid Ethers / Q000737:chemistry; D017433:Protein Structure, Secondary
chemical_list: C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D008565:Membrane Proteins; D008823:Micelles; D010742:Phospholipid Ethers