Kinetics of conformational transitions in cardiac troponin induced by Ca2+ dissociation determined by Förster resonance energy transfer.
Upon Ca2+ activation of cardiac muscle, several structural changes occur in the troponin subunits. These changes include the opening of the cardiac troponin C (cTnC) N-domain, the change of secondary structure of the inhibitory region of cardiac troponin I (cTnI), and the change in the separation between these two proteins in the cTnC-cTnI interface. We have used Förster resonance energy transfer in Ca2+ titration and stopped-flow experiments to delineate these transitions using a reconstituted cardiac troponin. Energy transfer results were quantified to yield time-dependent profiles of changes in intersite distances during Ca2+ dissociation. The closing of the cTnC N-domain induced by release of regulatory Ca2+ from cTnC occurs in one step (t1/2 approximately 5 ms), and this transition is not affected by Ca2+ release from the C-domain. The other two transitions triggered by Ca2+ dissociation are biphasic with the fast phase (t1/2 approximately 5 ms) correlated with Ca2+ release from the cTnC N-domain. These transitions are slower than the release of bound regulatory Ca2+ (t1/2 3.6 ms) and are coupled to one another in a cooperative manner in restoring their conformations in the deactivated state. The kinetic results define the magnitudes of structural changes relevant in Ca2+ switching between activation and deactivation of cardiac muscle contraction.
Dong
Wen-Ji
WJ
Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, AL 35294-0005, USA. wdong@uab.edu
Robinson
John M
JM
Xing
Jun
J
Cheung
Herbert C
HC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D004735:Energy Transfer; D007700:Kinetics; D051379:Mice; D009119:Muscle Contraction; D009154:Mutation; D009206:Myocardium / Q000737:chemistry; D011487:Protein Conformation; D021122:Protein Subunits / Q000235:genetics; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods*; D017186:Titrimetry; D014336:Troponin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D021122:Protein Subunits; D014336:Troponin; D002118:Calcium