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Three Distinct Actin-Attached Structural States of Myosin in Muscle Fibers
We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-l...
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Published in: | Biophysical journal 2012-03, Vol.102 (5), p.1088-1096 |
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description | We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M′.D.P, we used pPDM to cross-link SH1 (Cys707) to SH2 (Cys697) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M′.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M′D.P analog on the force generation pathway. |
doi_str_mv | 10.1016/j.bpj.2011.11.4027 |
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Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M′.D.P, we used pPDM to cross-link SH1 (Cys707) to SH2 (Cys697) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M′.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M′D.P analog on the force generation pathway.</description><identifier>ISSN: 0006-3495</identifier><identifier>EISSN: 1542-0086</identifier><identifier>DOI: 10.1016/j.bpj.2011.11.4027</identifier><identifier>PMID: 22404931</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Actins - metabolism ; active sites ; Animals ; Biochemistry ; Catalysis ; Catalytic Domain ; Cross-Linking Reagents - pharmacology ; crosslinking ; electron paramagnetic resonance spectroscopy ; Electron Spin Resonance Spectroscopy ; Electrons ; Free Radicals - chemistry ; Free Radicals - metabolism ; Maleimides - pharmacology ; muscle fibers ; Muscle Fibers, Skeletal - metabolism ; Muscle, Motility, and Motor Proteins ; Muscular system ; myosin ; Myosin Light Chains - chemistry ; Myosin Light Chains - metabolism ; Pyrroles - chemistry ; Pyrroles - metabolism ; Rabbits ; Stress, Mechanical ; Sulfhydryl Compounds - chemistry ; thiols</subject><ispartof>Biophysical journal, 2012-03, Vol.102 (5), p.1088-1096</ispartof><rights>2012 Biophysical Society</rights><rights>Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.</rights><rights>Copyright Biophysical Society Mar 7, 2012</rights><rights>2012 by the Biophysical Society. 2012 Biophysical Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c505t-f47e4190008b879050519514a5c66586c24fbdbe40ec640147decfdeaaf6df333</citedby><cites>FETCH-LOGICAL-c505t-f47e4190008b879050519514a5c66586c24fbdbe40ec640147decfdeaaf6df333</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3296056/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3296056/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22404931$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Mello, Ryan N.</creatorcontrib><creatorcontrib>Thomas, David D.</creatorcontrib><title>Three Distinct Actin-Attached Structural States of Myosin in Muscle Fibers</title><title>Biophysical journal</title><addtitle>Biophys J</addtitle><description>We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M′.D.P, we used pPDM to cross-link SH1 (Cys707) to SH2 (Cys697) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M′.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M′D.P analog on the force generation pathway.</description><subject>Actins - metabolism</subject><subject>active sites</subject><subject>Animals</subject><subject>Biochemistry</subject><subject>Catalysis</subject><subject>Catalytic Domain</subject><subject>Cross-Linking Reagents - pharmacology</subject><subject>crosslinking</subject><subject>electron paramagnetic resonance spectroscopy</subject><subject>Electron Spin Resonance Spectroscopy</subject><subject>Electrons</subject><subject>Free Radicals - chemistry</subject><subject>Free Radicals - metabolism</subject><subject>Maleimides - pharmacology</subject><subject>muscle fibers</subject><subject>Muscle Fibers, Skeletal - metabolism</subject><subject>Muscle, Motility, and Motor Proteins</subject><subject>Muscular system</subject><subject>myosin</subject><subject>Myosin Light Chains - chemistry</subject><subject>Myosin Light Chains - metabolism</subject><subject>Pyrroles - chemistry</subject><subject>Pyrroles - metabolism</subject><subject>Rabbits</subject><subject>Stress, Mechanical</subject><subject>Sulfhydryl Compounds - chemistry</subject><subject>thiols</subject><issn>0006-3495</issn><issn>1542-0086</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><recordid>eNp9kU1r3DAQhkVpaTZp_0APreklJ29Hn7ahFJa06QcJPSQ5C1keZ2W81kaSA_n3ldk0tD0UBkZIz7y8o5eQNxTWFKj6MKzb_bBmQOk6lwBWPSMrKgUrAWr1nKwAQJVcNPKIHMc4AFAmgb4kR4wJEA2nK_LjehsQi88uJjfZVGxs7uUmJWO32BVXKcw2zcGM-WgSxsL3xeWDj24qcl3O0Y5YnLsWQ3xFXvRmjPj6sZ-Qm_Mv12ffyoufX7-fbS5KK0GmshcVCtpkb3VbVw3kS9pIKoy0SslaWSb6tmtRAFolgIqqQ9t3aEyvup5zfkI-HXT3c7vDzuKUsj-9D25nwoP2xum_Xya31bf-XnPWKJAqC5w-CgR_N2NMeueixXE0E_o56oZVNV8sZvL9P-Tg5zDl7Rao4rymC8QOkA0-xoD9kxUKeglKDzoHpZegdK4lqDz09s8lnkZ-J5OBdwegN16b2-CivrnKEjKnSBlnyxofDwTmz753GHS0DieLnQtok-68-5-DX5YOrJM</recordid><startdate>20120307</startdate><enddate>20120307</enddate><creator>Mello, Ryan N.</creator><creator>Thomas, David D.</creator><general>Elsevier Inc</general><general>Biophysical Society</general><general>The Biophysical Society</general><scope>6I.</scope><scope>AAFTH</scope><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>7QP</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20120307</creationdate><title>Three Distinct Actin-Attached Structural States of Myosin in Muscle Fibers</title><author>Mello, Ryan N. ; Thomas, David D.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c505t-f47e4190008b879050519514a5c66586c24fbdbe40ec640147decfdeaaf6df333</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Actins - metabolism</topic><topic>active sites</topic><topic>Animals</topic><topic>Biochemistry</topic><topic>Catalysis</topic><topic>Catalytic Domain</topic><topic>Cross-Linking Reagents - pharmacology</topic><topic>crosslinking</topic><topic>electron paramagnetic resonance spectroscopy</topic><topic>Electron Spin Resonance Spectroscopy</topic><topic>Electrons</topic><topic>Free Radicals - chemistry</topic><topic>Free Radicals - metabolism</topic><topic>Maleimides - pharmacology</topic><topic>muscle fibers</topic><topic>Muscle Fibers, Skeletal - metabolism</topic><topic>Muscle, Motility, and Motor Proteins</topic><topic>Muscular system</topic><topic>myosin</topic><topic>Myosin Light Chains - chemistry</topic><topic>Myosin Light Chains - metabolism</topic><topic>Pyrroles - chemistry</topic><topic>Pyrroles - metabolism</topic><topic>Rabbits</topic><topic>Stress, Mechanical</topic><topic>Sulfhydryl Compounds - chemistry</topic><topic>thiols</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Mello, Ryan N.</creatorcontrib><creatorcontrib>Thomas, David D.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Biophysical journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mello, Ryan N.</au><au>Thomas, David D.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Three Distinct Actin-Attached Structural States of Myosin in Muscle Fibers</atitle><jtitle>Biophysical journal</jtitle><addtitle>Biophys J</addtitle><date>2012-03-07</date><risdate>2012</risdate><volume>102</volume><issue>5</issue><spage>1088</spage><epage>1096</epage><pages>1088-1096</pages><issn>0006-3495</issn><eissn>1542-0086</eissn><abstract>We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M′.D.P, we used pPDM to cross-link SH1 (Cys707) to SH2 (Cys697) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M′.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M′D.P analog on the force generation pathway.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>22404931</pmid><doi>10.1016/j.bpj.2011.11.4027</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Actins - metabolism active sites Animals Biochemistry Catalysis Catalytic Domain Cross-Linking Reagents - pharmacology crosslinking electron paramagnetic resonance spectroscopy Electron Spin Resonance Spectroscopy Electrons Free Radicals - chemistry Free Radicals - metabolism Maleimides - pharmacology muscle fibers Muscle Fibers, Skeletal - metabolism Muscle, Motility, and Motor Proteins Muscular system myosin Myosin Light Chains - chemistry Myosin Light Chains - metabolism Pyrroles - chemistry Pyrroles - metabolism Rabbits Stress, Mechanical Sulfhydryl Compounds - chemistry thiols |
title | Three Distinct Actin-Attached Structural States of Myosin in Muscle Fibers |
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