Monitoring shifts in the conformation equilibrium of the membrane protein cytochrome P450 reductase (POR) in nanodiscs

Maria Helena Wadsäter; Tomas Laursen; Aparajita Singha; Nikos Hatzakis; Dimitrios Stamou; Robert Barker; Kell Mortensen; Robert Krarup Feidenhans'l; Birger Lindberg Møller; Marite Cardenas Gomez

doi:10.1074/jbc.M112.400085

Monitoring shifts in the conformation equilibrium of the membrane protein cytochrome P450 reductase (POR) in nanodiscs

Maria Helena Wadsäter, Tomas Laursen, Aparajita Singha, Nikos Hatzakis, Dimitrios Stamou, Robert Barker, Kell Mortensen, Robert Krarup Feidenhans'l, Birger Lindberg Møller, Marite Cardenas Gomez

46 Citations (Scopus)

Abstract

Nanodiscs are self-assembled ∼50-nm² patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

Original language	English
Journal	Journal of Biological Chemistry
Volume	287
Issue number	41
Pages (from-to)	34596-34603
Number of pages	8
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M112.400085
Publication status	Published - 5 Oct 2012

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Wadsäter, M. H., Laursen, T., Singha, A., Hatzakis, N., Stamou, D., Barker, R., Mortensen, K., Feidenhans'l, R. K., Møller, B. L., & Cardenas Gomez, M. (2012). Monitoring shifts in the conformation equilibrium of the membrane protein cytochrome P450 reductase (POR) in nanodiscs. Journal of Biological Chemistry, 287(41), 34596-34603. https://doi.org/10.1074/jbc.M112.400085

Wadsäter, MH, Laursen, T, Singha, A, Hatzakis, N, Stamou, D, Barker, R, Mortensen, K , Feidenhans'l, RK , Møller, BL & Cardenas Gomez, M 2012, 'Monitoring shifts in the conformation equilibrium of the membrane protein cytochrome P450 reductase (POR) in nanodiscs', Journal of Biological Chemistry, vol. 287, no. 41, pp. 34596-34603. https://doi.org/10.1074/jbc.M112.400085

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abstract = "Nanodiscs are self-assembled ∼50-nm2 patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 {\AA}, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.",

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AU - Wadsäter, Maria Helena

AU - Laursen, Tomas

AU - Singha, Aparajita

AU - Hatzakis, Nikos

AU - Stamou, Dimitrios

AU - Barker, Robert

AU - Mortensen, Kell

AU - Feidenhans'l, Robert Krarup

AU - Møller, Birger Lindberg

AU - Cardenas Gomez, Marite

PY - 2012/10/5

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N2 - Nanodiscs are self-assembled ∼50-nm2 patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

AB - Nanodiscs are self-assembled ∼50-nm2 patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

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VL - 287

SP - 34596

EP - 34603

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 41

ER -

Monitoring shifts in the conformation equilibrium of the membrane protein cytochrome P450 reductase (POR) in nanodiscs

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