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
Abstract 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 i...
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| Format: | Article |
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Nature Portfolio
2017-06-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-017-04219-1 |
| _version_ | 1818994271852691456 |
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| author | Kazutoshi Yamamoto Marc A. Caporini Sang-Choul Im Lucy Waskell Ayyalusamy Ramamoorthy |
| author_facet | Kazutoshi Yamamoto Marc A. Caporini Sang-Choul Im Lucy Waskell Ayyalusamy Ramamoorthy |
| author_sort | Kazutoshi Yamamoto |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-12-20T20:55:18Z |
| format | Article |
| id | doaj.art-d6fc88d914294a2a968b2df38f418938 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-20T20:55:18Z |
| publishDate | 2017-06-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-d6fc88d914294a2a968b2df38f4189382022-12-21T19:26:51ZengNature PortfolioScientific Reports2045-23222017-06-017111310.1038/s41598-017-04219-1Transmembrane Interactions of Full-length Mammalian Bitopic Cytochrome-P450-Cytochrome-b5 Complex in Lipid Bilayers Revealed by Sensitivity-Enhanced Dynamic Nuclear Polarization Solid-state NMR SpectroscopyKazutoshi Yamamoto0Marc A. Caporini1Sang-Choul Im2Lucy Waskell3Ayyalusamy Ramamoorthy4Biophysics and Department of Chemistry, University of MichiganBruker Biospin Corporation, 15 Fortune DriveDepartment of Anesthesiology, VA Medical Center, University of MichiganDepartment of Anesthesiology, VA Medical Center, University of MichiganBiophysics and Department of Chemistry, University of MichiganAbstract 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.https://doi.org/10.1038/s41598-017-04219-1 |
| spellingShingle | Kazutoshi Yamamoto Marc A. Caporini Sang-Choul Im Lucy Waskell Ayyalusamy Ramamoorthy 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 Scientific Reports |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| url | https://doi.org/10.1038/s41598-017-04219-1 |
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