Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton-detected solid-state NMR
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ARTICLE
Resonance assignment of the outer membrane protein AlkL in lipid bilayers by proton‑detected solid‑state NMR Tobias Schubeis1 · Tom S. Schwarzer2 · Tanguy Le Marchand1 · Jan Stanek1 · Kumar Tekwani Movellan3 · Kathrin Castiglione2,4 · Guido Pintacuda1 · Loren B. Andreas1,3 Received: 16 April 2020 / Accepted: 19 June 2020 © Springer Nature B.V. 2020
Abstract Most commonly small outer membrane proteins, possessing between 8 and 12 β-strands, are not involved in transport but fulfill diverse functions such as cell adhesion or binding of ligands. An intriguing exception are the 8-stranded β-barrel proteins of the OmpW family, which are implicated in the transport of small molecules. A representative example is AlkL from Pseudomonas putida GPoI, which functions as a passive importer of hydrophobic molecules. This role is of high interest with respect to both fundamental biological understanding and industrial applications in biocatalysis, since this protein is frequently utilized in biotransformation of alkanes. While the transport function of AlkL is generally accepted, a controversy in the transport mechanism still exists. In order to address this, we are pursuing a structural study of recombinantly produced AlkL reconstituted in lipid bilayers using solid-state NMR spectroscopy. In this manuscript we present 1H, 13C and 15N chemical shift assignments obtained via a suite of 3D experiments employing high magnetic fields (1 GHz and 800 MHz) and the latest magic-angle spinning (MAS) approaches at fast (60–111) kHz rates. We additionally analyze the secondary structure prediction in comparison with those of published structures of homologous proteins. Keywords 1H-detected solid-state NMR · Membrane proteins · Lipid bilayers · Alkane transport · Beta-barrel
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12104-020-09964-5) contains supplementary material, which is available to authorized users.
OG N-octyl-β-d-glucopyranoside DMPC 1,2-Dimyristoyl-sn-glycero-3-phosphocholine CP Cross polarization INEPT Insensitive nuclei enhanced by polarization transfer BASS-SD Band-selective spectral spin diffusion TOCSY Total correlation spectroscopy MAS Magic-angle spinning
* Guido Pintacuda guido.pintacuda@ens‑lyon.fr
Biological Context
Abbreviations IMAC Immobilized metal affinity chromatography EDTA Ethylenediaminetetraacetic acid LDAO Lauryldimethylamine N-oxide
* Loren B. Andreas [email protected] 1
Centre de RMN à Très Hauts Champs de Lyon (FRE 2034 CNRS, UCB Lyon 1, ENS Lyon), Université de Lyon, 5 rue de la Doua, 69100 Villeurbanne, France
2
Institute of Biochemical Engineering, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, Germany
3
Department for NMR‑Based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany
4
Present Address: Institute of Bioprocess Engineering, FAU Erlangen-Nürnberg, Paul‑Gordan Str. 3, 91052 Erlangen, Germany
Due to the ability
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