The cardiac Ca(2+)-deficient EF-hand governs the phenotype of the cardiac-skeletal TnC-chimera in solution by Sr(2+)-induced tryptophan fluorescence emission.
In the development of force during Sr2+ activation, phenotypically cardiac muscle is more sensitive than fast-twitch skeletal muscle, and TnC is central in this mechanism. The uncertainty has remained, however, whether such functional manifestations in situ relied critically on protein-protein interactions in the fiber or whether the Sr2+ sensitivities were governed intrinsically within the TnC molecule. To resolve this, we substituted a tryptophan for phenylalanine-26 in both rabbit sTnC (sTnC.W26) and in a chimera (c1/s.W26) where the 41 N-terminal amino acid residues were of bovine cTnC and the remaining 42-160 residues of rabbit sTnC. The metal ion dependent fluorescence emissions of the constructs could be examined in solution isolated from the protein-protein interactions found in situ. The Sr2+ sensitivities of these proteins differed by 0.55 +/- 0.02 pSr unit, but Ca2+ sensitivities were indistinguishable, as in the fiber. In another mutant, where the 27VLGA30 cluster was replaced with D-AD to enable site 1 to coordinate metal ion binding despite closely preserved cardiac structure, the Sr(2+)-sensitivity response was transformed into the skeletal-type. The Hill coefficients were also characteristically distinct for the various constructs. The findings indicate that cardiac N-terminal 41 residues define TnC performance in solution similar to that in situ. Moreover, the study provides unambiguous evidence that TnC isoforms intrinsically dominate the phenotype in the switching mechanisms for both cardiac and skeletal contractilities.
Gulati
J
J
Department of Physiology/Biophysics and Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
Rao
V G
VG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D006321:Heart / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology; D009200:Myocardial Contraction; D011487:Protein Conformation / Q000187:drug effects*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013324:Strontium / Q000494:pharmacology*; D014336:Troponin / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D019209:Troponin C; D014364:Tryptophan
D011993:Recombinant Fusion Proteins; D012996:Solutions; D014336:Troponin; D019209:Troponin C; D014364:Tryptophan; D002118:Calcium; D013324:Strontium