The dynamic protein-protein and protein-ligand interactions of integral bitopic membrane proteins with a single membrane-spanning helix play a plethora of vital roles in the cellular processes associated with human health and diseases, including signaling and enzymatic catalysis. While an increasing number of high-resolution structural studies of membrane proteins have successfully manifested an in-depth understanding of their biological functions, intact membrane-bound bitopic protein-protein complexes pose tremendous challenges for structural studies by crystallography or solution NMR spectroscopy. Therefore, there is a growing interest in developing approaches to investigate the functional interactions of bitopic membrane proteins embedded in lipid bilayers at atomic-level. Here we demonstrate the feasibility of dynamic nuclear polarization (DNP) magic-angle-spinning NMR techniques, along with a judiciously designed stable isotope labeling scheme, to measure atomistic-resolution transmembrane-transmembrane interactions of full-length mammalian ~72-kDa cytochrome P450-cytochrome b5 complex in lipid bilayers. Additionally, the DNP sensitivity-enhanced two-dimensional 13C/13C chemical shift correlations via proton driven spin diffusion provided distance constraints to characterize protein-lipid interactions and revealed the transmembrane topology of cytochrome b5. The results reported in this study would pave ways for high-resolution structural and topological investigations of membrane-bound full-length bitopic protein complexes under physiological conditions.
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[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] Solid-state NMR studies of full-length BamA in lipid bilayers suggest limited overall POTRA mobility.
Solid-state NMR studies of full-length BamA in lipid bilayers suggest limited overall POTRA mobility.
Related Articles Solid-state NMR studies of full-length BamA in lipid bilayers suggest limited overall POTRA mobility.
J Mol Biol. 2014 Feb 12;
Authors: Sinnige T, Weingarth M, Renault M, Baker L, Tommassen J, Baldus M
Abstract
The outer membrane protein BamA is the key player in ?-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and...
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02-19-2014 12:07 AM
[NMR paper] Probing the transmembrane structure and topology of microsomal cytochrome-p450 by solid-state NMR on temperature-resistant bicelles.
Probing the transmembrane structure and topology of microsomal cytochrome-p450 by solid-state NMR on temperature-resistant bicelles.
Probing the transmembrane structure and topology of microsomal cytochrome-p450 by solid-state NMR on temperature-resistant bicelles.
Sci Rep. 2013 Aug 30;3:2556
Authors: Yamamoto K, Gildenberg M, Ahuja S, Im SC, Pearcy P, Waskell L, Ramamoorthy A
Abstract
Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the...
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08-31-2013 06:56 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
[NMR paper] Substrate-Modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR.
Substrate-Modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR.
Related Articles Substrate-Modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR.
J Biol Chem. 2013 Apr 25;
Authors: Estrada DF, Laurence JS, Scott EE
Abstract
The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Herein the interactions between the...
Transmembrane Interactions of Full-length Mammalian Bitopic Cytochrome-P450-Cytochrome-b5 Complex in Lipid Bilayers Revealed by Sensitivity-Enhanced Dynamic Nuclear Polarization Solid-state NMR Spectroscopy #DNPNMR
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