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Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b5 and Cytochrome c
Cytochrome b 5 (cyt b 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 cyt b 5 a...
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Published in: | Scientific reports 2017-08, Vol.7 (1) |
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creator | Gentry, Katherine A. Prade, Elke Barnaba, Carlo Zhang, Meng Mahajan, Mukesh Im, Sang-Choul Anantharamaiah, G. M. Nagao, Satoshi Waskell, Lucy Ramamoorthy, Ayyalusamy |
description | Cytochrome
b
5
(cyt
b
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 cyt
b
5
and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cyt
b
5
- cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cyt
b
5
-cyt c binding interface. Our experimental results identify differences in the binding epitope of cyt
b
5
in the presence and absence of membrane. Notably, in the presence of membrane, cyt
b
5
only engaged cyt c at its lower and upper clefts while the membrane-free cyt
b
5
also uses a distal region. Using restraints generated from both cyt
b
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. |
doi_str_mv | 10.1038/s41598-017-08130-7 |
format | article |
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b
5
(cyt
b
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 cyt
b
5
and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cyt
b
5
- cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cyt
b
5
-cyt c binding interface. Our experimental results identify differences in the binding epitope of cyt
b
5
in the presence and absence of membrane. Notably, in the presence of membrane, cyt
b
5
only engaged cyt c at its lower and upper clefts while the membrane-free cyt
b
5
also uses a distal region. Using restraints generated from both cyt
b
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.</description><identifier>EISSN: 2045-2322</identifier><identifier>DOI: 10.1038/s41598-017-08130-7</identifier><identifier>PMID: 28798301</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>101/6 ; 140/131 ; 631/57/2271 ; 639/638/45/535/878/1263 ; 82 ; Humanities and Social Sciences ; multidisciplinary ; Science ; Science (multidisciplinary)</subject><ispartof>Scientific reports, 2017-08, Vol.7 (1)</ispartof><rights>The Author(s) 2017</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-p184t-b481046f27a01f460f6e00e6cf4f894c7031adf711a165b60d680e6c4381259a3</cites><orcidid>0000-0002-8153-7613</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552742/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552742/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids></links><search><creatorcontrib>Gentry, Katherine A.</creatorcontrib><creatorcontrib>Prade, Elke</creatorcontrib><creatorcontrib>Barnaba, Carlo</creatorcontrib><creatorcontrib>Zhang, Meng</creatorcontrib><creatorcontrib>Mahajan, Mukesh</creatorcontrib><creatorcontrib>Im, Sang-Choul</creatorcontrib><creatorcontrib>Anantharamaiah, G. M.</creatorcontrib><creatorcontrib>Nagao, Satoshi</creatorcontrib><creatorcontrib>Waskell, Lucy</creatorcontrib><creatorcontrib>Ramamoorthy, Ayyalusamy</creatorcontrib><title>Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b5 and Cytochrome c</title><title>Scientific reports</title><addtitle>Sci Rep</addtitle><description>Cytochrome
b
5
(cyt
b
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 cyt
b
5
and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cyt
b
5
- cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cyt
b
5
-cyt c binding interface. Our experimental results identify differences in the binding epitope of cyt
b
5
in the presence and absence of membrane. Notably, in the presence of membrane, cyt
b
5
only engaged cyt c at its lower and upper clefts while the membrane-free cyt
b
5
also uses a distal region. Using restraints generated from both cyt
b
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.</description><subject>101/6</subject><subject>140/131</subject><subject>631/57/2271</subject><subject>639/638/45/535/878/1263</subject><subject>82</subject><subject>Humanities and Social Sciences</subject><subject>multidisciplinary</subject><subject>Science</subject><subject>Science (multidisciplinary)</subject><issn>2045-2322</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><recordid>eNpVkMtOwzAQRS0kRKvSH2DlHwj4GTsbJBSVhyhiAawtx7GbVI1dOQ6ifD1JywK8GXmO5mrmAHCF0TVGVN70DPNCZgiLDElMUSbOwJwgxjNCCZmBZd9v0fg4KRguLsCMSFFIivAc9M-tt6k1UPsavqU4mDREvYNlo6M2ycb2W6c2eBgcTI2FK-esSf30fbFdFbW3cKQTKkO339mvCZWHFEwTQ2dhxY_RfzrmEpw7vevt8rcuwMf96r18zNavD0_l3TrbY8lSVjGJEcsdERphx3LkcouQzY1jThbMCESxrp3AWOOcVzmqczlhRiUmvNB0AW5Pufuh6mxtrE_jaWof207Hgwq6Vf-Jbxu1CZ-Kc04EI2MAPQX044zf2Ki2YYh-3FlhpCb16qRejerVUb0S9AeOBXjk</recordid><startdate>20170810</startdate><enddate>20170810</enddate><creator>Gentry, Katherine A.</creator><creator>Prade, Elke</creator><creator>Barnaba, Carlo</creator><creator>Zhang, Meng</creator><creator>Mahajan, Mukesh</creator><creator>Im, Sang-Choul</creator><creator>Anantharamaiah, G. M.</creator><creator>Nagao, Satoshi</creator><creator>Waskell, Lucy</creator><creator>Ramamoorthy, Ayyalusamy</creator><general>Nature Publishing Group UK</general><scope>C6C</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-8153-7613</orcidid></search><sort><creationdate>20170810</creationdate><title>Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b5 and Cytochrome c</title><author>Gentry, Katherine A. ; Prade, Elke ; Barnaba, Carlo ; Zhang, Meng ; Mahajan, Mukesh ; Im, Sang-Choul ; Anantharamaiah, G. M. ; Nagao, Satoshi ; Waskell, Lucy ; Ramamoorthy, Ayyalusamy</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p184t-b481046f27a01f460f6e00e6cf4f894c7031adf711a165b60d680e6c4381259a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>101/6</topic><topic>140/131</topic><topic>631/57/2271</topic><topic>639/638/45/535/878/1263</topic><topic>82</topic><topic>Humanities and Social Sciences</topic><topic>multidisciplinary</topic><topic>Science</topic><topic>Science (multidisciplinary)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gentry, Katherine A.</creatorcontrib><creatorcontrib>Prade, Elke</creatorcontrib><creatorcontrib>Barnaba, Carlo</creatorcontrib><creatorcontrib>Zhang, Meng</creatorcontrib><creatorcontrib>Mahajan, Mukesh</creatorcontrib><creatorcontrib>Im, Sang-Choul</creatorcontrib><creatorcontrib>Anantharamaiah, G. M.</creatorcontrib><creatorcontrib>Nagao, Satoshi</creatorcontrib><creatorcontrib>Waskell, Lucy</creatorcontrib><creatorcontrib>Ramamoorthy, Ayyalusamy</creatorcontrib><collection>Springer Nature OA Free Journals</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Scientific reports</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gentry, Katherine A.</au><au>Prade, Elke</au><au>Barnaba, Carlo</au><au>Zhang, Meng</au><au>Mahajan, Mukesh</au><au>Im, Sang-Choul</au><au>Anantharamaiah, G. M.</au><au>Nagao, Satoshi</au><au>Waskell, Lucy</au><au>Ramamoorthy, Ayyalusamy</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b5 and Cytochrome c</atitle><jtitle>Scientific reports</jtitle><stitle>Sci Rep</stitle><date>2017-08-10</date><risdate>2017</risdate><volume>7</volume><issue>1</issue><eissn>2045-2322</eissn><abstract>Cytochrome
b
5
(cyt
b
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 cyt
b
5
and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cyt
b
5
- cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cyt
b
5
-cyt c binding interface. Our experimental results identify differences in the binding epitope of cyt
b
5
in the presence and absence of membrane. Notably, in the presence of membrane, cyt
b
5
only engaged cyt c at its lower and upper clefts while the membrane-free cyt
b
5
also uses a distal region. Using restraints generated from both cyt
b
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.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>28798301</pmid><doi>10.1038/s41598-017-08130-7</doi><orcidid>https://orcid.org/0000-0002-8153-7613</orcidid><oa>free_for_read</oa></addata></record> |
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source | Publicly Available Content Database; PubMed Central; Free Full-Text Journals in Chemistry; Springer Nature - nature.com Journals - Fully Open Access |
subjects | 101/6 140/131 631/57/2271 639/638/45/535/878/1263 82 Humanities and Social Sciences multidisciplinary Science Science (multidisciplinary) |
title | Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b5 and Cytochrome c |
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