The Cardiac Ca2+-Deficient EF-Hand Governs the Phenotype of the Cardiac-Skeletal TnC-Chimera in Solution by Sr2+-Induced Tryptophan Fluorescence Emission

Abstract

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.

Disparate Fluorescence Properties of 2-[4′-(Iodoacetamido)anilino]-Naphthalene-6-Sulfonic Acid Attached to Cys-84 and Cys-35 of Troponin C in Cardiac Muscle Troponin

“…In a similar chimeric TnC in which the inactive Ca2+ binding loop I was activated to chelate Ca2+ by genetic modification, the cardiac-type Sr2+ sensitivity was found to transform into the skeletal-type (Gulati and Rao, 1994). Taken together, the present results and those of previous studies strongly suggest that flexible loop I, in which the side chains are not coordinated to a metal ion, may be essential as part of the trigger mechanism in cardiac muscle.…”

Ca2+binding to cardiac troponin C: effects of temperature and pH on mammalian and salmonid isoforms

“…It is suggested that this low-temperature effect could increase the activation energy required to trigger the conformational response (42). Full-length mammalian sTnC is 66.7% identical in amino acid sequence to mammalian cTnC, and the two isoforms display distinct functional differences (1,17,18,20,39) and differ in the number of functional Ca 2ϩ binding sites. For these reasons, prediction of how lowered environmental temperature would affect the structure of mammalian cTnC from data on sTnC is tenuous.…”

Molecular Mobility of the Ca2+-Deficient EF-Hand of Cardiac Troponin as Revealed by Fluorescence Polarization of Genetically Inserted Tryptophan

“…Both the normal and tryptophan 26 derivatives of skeletal TnC (sTnC4 and sTnC4.W), the cardiac/skeletal chimera with Ca2+-deficient site I (cl/s and cl/s.W), and the Ca2+binding variants (CBcl/s and CBcl/s.W) were the same as before (Gulati & Rao, 1994). The tryptophan derivative of sTnC-, an inactive skeletal TnC in which site I was made Ca2+-deficient by replacing the putative x-coordinate in the skeletal EF-hand [see Kretsinger (1980)] from Asp27-* Ala, was generated especially for this study.…”

“…The codon for tryptophan was inserted in position 26 by replacing that for phenylalanine, exactly as in the other constructs. Because no tryptophan existed in bovine cTnC or rabbit sTnC, Trp26 provided a ready spectroscopic marker that could be followed with ease (Gulati & Rao, 1994). The nucleotide sequences of the mutants were verified by DNA sequencing.…”

“…Recent insights into the mechanistic aspects of the cardiac Ca2+ switch for contractile activation were inferred from steady-state fluorescence emissions by the excited tryptophan (Trp26) in the Ca2+-deficient region (site I) of a modified cardiac/skeletal chimera (Gulati & Rao, 1994). The quantum yield of the tryptophan was enhanced in saturating Ca2+, suggesting that the site I region was active when the switch was turned on.…”