Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c

Abstract Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cy...

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Autores principales:	Katherine A. Gentry, Elke Prade, Carlo Barnaba, Meng Zhang, Mukesh Mahajan, Sang-Choul Im, G. M. Anantharamaiah, Satoshi Nagao, Lucy Waskell, Ayyalusamy Ramamoorthy
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/25723ddda3f34b8b832c8fae21511602
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spelling	oai:doaj.org-article:25723ddda3f34b8b832c8fae215116022021-12-02T12:32:20ZKinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c10.1038/s41598-017-08130-72045-2322https://doaj.org/article/25723ddda3f34b8b832c8fae215116022017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-08130-7https://doaj.org/toc/2045-2322Abstract Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb 5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb 5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb 5-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb 5 in the presence and absence of membrane. Notably, in the presence of membrane, cytb 5 only engaged cyt c at its lower and upper clefts while the membrane-free cytb 5 also uses a distal region. Using restraints generated from both cytb 5 and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. These results demonstrate the importance of studying protein-protein complex formation in membrane mimetic systems. Our results also demonstrate the successful preparation of novel peptide-based lipid nanodiscs, which are detergent-free and possesses size flexibility, and their use for NMR structural studies of membrane proteins.Katherine A. GentryElke PradeCarlo BarnabaMeng ZhangMukesh MahajanSang-Choul ImG. M. AnantharamaiahSatoshi NagaoLucy WaskellAyyalusamy RamamoorthyNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-15 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Katherine A. Gentry Elke Prade Carlo Barnaba Meng Zhang Mukesh Mahajan Sang-Choul Im G. M. Anantharamaiah Satoshi Nagao Lucy Waskell Ayyalusamy Ramamoorthy Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c
description	Abstract Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb 5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb 5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb 5-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb 5 in the presence and absence of membrane. Notably, in the presence of membrane, cytb 5 only engaged cyt c at its lower and upper clefts while the membrane-free cytb 5 also uses a distal region. Using restraints generated from both cytb 5 and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. These results demonstrate the importance of studying protein-protein complex formation in membrane mimetic systems. Our results also demonstrate the successful preparation of novel peptide-based lipid nanodiscs, which are detergent-free and possesses size flexibility, and their use for NMR structural studies of membrane proteins.
format	article
author	Katherine A. Gentry Elke Prade Carlo Barnaba Meng Zhang Mukesh Mahajan Sang-Choul Im G. M. Anantharamaiah Satoshi Nagao Lucy Waskell Ayyalusamy Ramamoorthy
author_facet	Katherine A. Gentry Elke Prade Carlo Barnaba Meng Zhang Mukesh Mahajan Sang-Choul Im G. M. Anantharamaiah Satoshi Nagao Lucy Waskell Ayyalusamy Ramamoorthy
author_sort	Katherine A. Gentry
title	Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c
title_short	Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c
title_full	Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c
title_fullStr	Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c
title_full_unstemmed	Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c
title_sort	kinetic and structural characterization of the effects of membrane on the complex of cytochrome b 5 and cytochrome c
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/25723ddda3f34b8b832c8fae21511602
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Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c

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