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Application of methyl-TROSY to a large paramagnetic membrane protein without perdeuteration: 13C-MMTS-labeled NADPH-cytochrome P450 oxidoreductase

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Abstract

NMR spectroscopy of membrane proteins involved in electron transport is difficult due to the presence of both the lipids and paramagnetic centers. Here we report the solution NMR study of the NADPH-cytochrome P450 oxidoreductase (POR) in its reduced and oxidized states. We interrogate POR, first, in its truncated soluble form (70 kDa), which is followed by experiments with the full-length protein incorporated in a lipid nanodisc (240 kDa). To overcome paramagnetic relaxation in the reduced state of POR as well as the signal broadening due to its high molecular weight, we utilized the methyl-TROSY approach. Extrinsic 13C-methyl groups were introduced by modifying the engineered surface-exposed cysteines with methyl-methanethiosulfonate. Chemical shift dispersion of the resonances from different sites in POR was sufficient to monitor differential effects of the reduction–oxidation process and conformation changes in the POR structure related to its function. Despite the high molecular weight of the POR-nanodisc complex, the surface-localized 13C-methyl probes were sufficiently mobile to allow for signal detection at 600 MHz without perdeuteration. This work demonstrates a potential of the solution methyl-TROSY in analysis of structure, dynamics, and function of POR, which may also be applicable to similar paramagnetic and flexible membrane proteins.

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Acknowledgements

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R15 GM126528-01 to ELK and by R01 GM097031 to JJK, and also—by the Regular Research Grant 2016 from Committee on Research (COR), Marquette University. ARG acknowledges Eisch Research Fellowship during the academic year 2016–2017. Authors acknowledge NMR instrument center of Biochemistry Department of the Medical College of Wisconsin for providing access to the Bruker 600 MHz instrument. The authors are grateful to Dr. Blake Hill (Medical College of Wisconsin, Milwaukee) and Dr. Kevin MacKenzie (Baylor College of Medicine, Houston) for acquisition of high-field NMR data on the POR-nanodisc sample at the NMR facility of the Baylor College of Medicine.

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Authors and Affiliations

  1. Chemistry Department, Marquette University, Milwaukee, WI, USA

    Azamat R. Galiakhmetov & Evgenii L. Kovrigin

  2. Biochemistry Department, Medical College of Wisconsin, Milwaukee, WI, USA

    Elizaveta A. Kovrigina, Chuanwu Xia & Jung-Ja P. Kim

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  1. Azamat R. Galiakhmetov
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  2. Elizaveta A. Kovrigina
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  3. Chuanwu Xia
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  5. Evgenii L. Kovrigin
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Correspondence to Jung-Ja P. Kim or Evgenii L. Kovrigin.

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Galiakhmetov, A.R., Kovrigina, E.A., Xia, C. et al. Application of methyl-TROSY to a large paramagnetic membrane protein without perdeuteration: 13C-MMTS-labeled NADPH-cytochrome P450 oxidoreductase. J Biomol NMR 70, 21–31 (2018). https://doi.org/10.1007/s10858-017-0152-3

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