Ca2+ - and cross-bridge-dependent changes in N- and C-terminal structure of troponin C in rat cardiac muscle.
Linear dichroism of 5'-tetramethylrhodamine (5'ATR)-labeled cardiac troponin C (cTnC) was measured to monitor cTnC structure during Ca2+-activation of force in rat skinned myocardium. Mono-cysteine mutants allowed labeling at Cys-84 (cTnC(C84), near the D/E helix linker); Cys-35 (cTnC(C35), at nonfunctional site I); or near the C-terminus with a cysteine inserted at site 98 (cTnC-C35S,C84S,S98C, cTnC(C98)). With 5'ATR-labeled cTnC(C84) and cTnC(C98) dichroism increased with increasing [Ca2+], while rigor cross-bridges caused dichroism to increase more with 5'ATR-labeled cTnC(C84) than cTnC(C98). The pCa50 values and n(H) from Hill analysis of the Ca2+-dependence of force and dichroism were 6.4 (+/-0.02) and 1.08 (+/-0.04) for force and 6.3 (+/-0.04) and 1.02 (+/-0.09) (n = 5) for dichroism in cTnC(C84) reconstituted trabeculae. Corresponding data from cTnC(C98) reconstituted trabeculae were 5.53 (+/-0.03) and 3.1 (+/-0.17) for force, and 5.39 (+/-0.03) and 1.87 (+/-0.17) (n = 5) for dichroism. The contribution of active cycling cross-bridges to changes in cTnC structure was determined by inhibition of force to 6% of pCa 4.0 controls with 1.0 mM sodium vanadate (Vi). With 5'ATR-labeled cTnC(C84) Vi caused both the pCa50)of dichroism and the maximum value at pCa 4.0 to decrease, while with 5'ATR-labeled cTnC(C98) the pCa50 of dichroism decreased with no change of dichroism at pCa 4.0. The dichroism of 5'ATR-labeled cTnC(C35) was insensitive to either Ca2+ or strong cross-bridges. These data suggest that both Ca2+ and cycling cross-bridges perturb the N-terminal structure of cTnC at Cys-84, while C-terminal structure is altered by site II Ca2+-binding, but not cross-bridges.
Martyn
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Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA. dmartyn@u.washington.edu
Regnier
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Xu
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Gordon
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D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008297:Male; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D012235:Rhodamines; D019209:Troponin C; C034996:tetramethylrhodamine iodoacetamide; D003545:Cysteine; D002118:Calcium