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 (70Â*kDa), which is followed by experiments with the full-length protein incorporated in a lipid nanodisc (240Â*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 reductionâ??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 600Â*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.
CH3] Labeled Proteins for Methyl-TROSY NMR - SelectScience
CH3] Labeled Proteins for Methyl-TROSY NMR - SelectScience
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CH3] Labeled Proteins for Methyl-TROSY NMR
SelectScience
Isotope labelling has revolutionized the use of biomolecular NMR spectroscopy, allowing the exploration of molecular interactions with high sensitivity and resolution. Introducing labelled Thr, a protein often found at molecular interfaces and involved ...
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07-19-2017 01:32 PM
CH3] Labeled Proteins for Methyl-TROSY NMR - SelectScience
CH3] Labeled Proteins for Methyl-TROSY NMR - SelectScience
<img alt="" height="1" width="1">
CH3] Labeled Proteins for Methyl-TROSY NMR
SelectScience
Isotope labelling has revolutionized the use of biomolecular NMR spectroscopy, allowing the exploration of molecular interactions with high sensitivity and resolution. Introducing labelled Thr, a protein often found at molecular interfaces and involved ...
and more »
Read here
nmrlearner
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07-17-2017 04:06 AM
CH3] Labeled Proteins for Methyl-TROSY NMR - SelectScience
CH3] Labeled Proteins for Methyl-TROSY NMR - SelectScience
<img alt="" height="1" width="1">
CH3] Labeled Proteins for Methyl-TROSY NMR
SelectScience
Isotope labelling has revolutionized the use of biomolecular NMR spectroscopy, allowing the exploration of molecular interactions with high sensitivity and resolution. Introducing labelled Thr, a protein often found at molecular interfaces and involved ...
Read here
nmrlearner
Online News
0
07-14-2017 01:32 AM
[NMR paper] Effects of Membrane Mimetics on Cytochrome P450-Cytochrome b5 Interactions Characterized by NMR Spectroscopy.
Effects of Membrane Mimetics on Cytochrome P450-Cytochrome b5 Interactions Characterized by NMR Spectroscopy.
Effects of Membrane Mimetics on Cytochrome P450-Cytochrome b5 Interactions Characterized by NMR Spectroscopy.
J Biol Chem. 2015 Mar 20;
Authors: Zhang M, Huang R, Im SC, Waskell L, Ramamoorthy A
Abstract
Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450...
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03-22-2015 06:36 PM
[NMR paper] Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR.
Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR.
Related Articles Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR.
Biophys J. 2014 May 20;106(10):2126-33
Authors: Huang R, Yamamoto K, Zhang M, Popovych N, Hung I, Im SC, Gan Z, Waskell L, Ramamoorthy A
Abstract
NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of...
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05-24-2014 04:50 PM
[NMR paper] Dynamic Interaction Between Membrane-Bound Full-Length Cytochrome P450 and Cytochrome b5 Observed by Solid-State NMR Spectroscopy.
Dynamic Interaction Between Membrane-Bound Full-Length Cytochrome P450 and Cytochrome b5 Observed by Solid-State NMR Spectroscopy.
Dynamic Interaction Between Membrane-Bound Full-Length Cytochrome P450 and Cytochrome b5 Observed by Solid-State NMR Spectroscopy.
Sci Rep. 2013 Aug 29;3:2538
Authors: Yamamoto K, Dürr UH, Xu J, Im SC, Waskell L, Ramamoorthy A
Abstract
Microsomal monoxygenase enzymes of the cytochrome-P450 family are found in all biological kingdoms, and play a central role in the breakdown of metabolic as well as...
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08-30-2013 04:35 PM
[NMR paper] A model of the membrane-bound cytochrome b5-cytochrome p450 complex from NMR and mutagenesis data.
A model of the membrane-bound cytochrome b5-cytochrome p450 complex from NMR and mutagenesis data.
A model of the membrane-bound cytochrome b5-cytochrome p450 complex from NMR and mutagenesis data.
J Biol Chem. 2013 May 24;
Authors: Ahuja S, Jahr N, Im SC, Vivekanandan S, Popovych N, Le Clair SV, Huang R, Soong R, Xu J, Yamamoto K, Nanga RP, Bridges A, Waskell L, Ramamoorthy A
Abstract
Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450)....
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05-28-2013 06:36 PM
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methylâ??methyl nuclear overhauser enhancement spectroscopy
Abstract Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic...
Application of methyl-TROSY to a large paramagnetic membrane protein without perdeuteration: 13 C-MMTS-labeled NADPH-cytochrome P450 oxidoreductase
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