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 Citationer (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.

Originalsprog	Engelsk
Tidsskrift	Journal of Biological Chemistry
Vol/bind	287
Udgave nummer	41
Sider (fra-til)	34596-34603
Antal sider	8
ISSN	0021-9258
DOI	https://doi.org/10.1074/jbc.M112.400085
Status	Udgivet - 5 okt. 2012

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10.1074/jbc.M112.400085

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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, bind 287, nr. 41, s. 34596-34603. https://doi.org/10.1074/jbc.M112.400085

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

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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DO - 10.1074/jbc.M112.400085

M3 - Journal article

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SN - 0021-9258

VL - 287

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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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