Site-directed mutation of the trigger calcium-binding sites in cardiac troponin C.
The trigger Ca2+-binding sites in troponin C, those which initiate muscle contraction, are thought to be the first two of four potential sites (sites I-IV). In cardiac troponin C, the first Ca2+-binding site is inactive, and initiation of contraction in cardiac muscle appears to involve only the second site. To study this phenomenon and associated Ca2+-dependent protein conformational changes in cardiac troponin C, the cDNA for the chicken protein was incorporated into a bacterial expression plasmid to allow site-specific mutagenesis. Ca2+-binding site I was activated by deletion of Val-28 and conversion of amino acids 29-32 to those found at the first four positions in the active site I of fast skeletal troponin C. In a series of proteins, Ca2+-binding site II was inactivated by mutation of amino acids Asp-65, Asp-67, and Gly-70. All mutated proteins exhibited the predicted calcium-binding characteristics. The single mutation of converting Asp-65 to Ala was sufficient to inactivate site II. Ca2+-dependent conformational changes in the normal and mutated proteins were monitored by labeling with a sulfhydryl-specific fluorescent dye. Activation of Ca2+-binding site I or inactivation of site II, eliminated the large Ca2+-dependent increase in fluorescence seen in the wild type protein and there was, instead, a Ca2+-dependent decrease in fluorescence. All mutant proteins could associate with troponin I and troponin T to form a troponin complex. Activation of Ca2+-binding site I changed the characteristics of contraction in skinned slow skeletal muscle fibers such that the response to Ca2+ was more cooperative. Inactivation of Ca2+-binding site II abolished Ca2+-dependent contraction in skinned muscle fibers. The data provide a direct demonstration that Ca2+-binding site II in cardiac troponin C is essential for triggering muscle contraction and support the hypothesis that site I functions to modify the characteristics of contraction.
Putkey
J A
JA
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Sweeney
H L
HL
Campbell
S T
ST
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D005260:Female; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D009132:Muscles / Q000502:physiology; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000235:genetics / Q000378:metabolism*; D019209:Troponin C
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D002118:Calcium