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J Gulati

Publications and source records attributed to J Gulati.

At least 19 recordsLinked to original sources
PubMed1998

A model for the function of the bisphosphorylated heart-specific troponin-I N-terminus.

Bisphosphorylation of two adjacently located serine residues in the heart-specific N-terminus of the cTnl subunit reduces calcium affinity of the cTnC subunit. An interaction of the phosphorylation region of cTnI with acidic residues of another cTn subunit has been proposed formerly based on 31P nuclear magnetic resonance (NMR) data. A possible candidate is cTnC. Thus, an interaction model of cTnC with the bisphosphorylated cTnI N-terminus has been built using a homology model of hcTnC based on the crystal structure of tusTnC and the structure of the phosphorylation region of cTnI determined by 2D NMR. By computational search, five cluster of acidic residues of cTnC might interact with the cTnI phosphorylation region. Three sites could be excluded by 31P-NMR experiments. The two remaining sites are located in the N-terminal helix of cTnC and between calcium binding sites III and IV. Reorientation of the arginine and phosphoserine sidechains within the phosphorylation region as proposed by refined docking could explain the formerly measured changes in pKaapp values. Thus, local pKa changes might lead to the reduction of calcium affinity observed upon cTnI bisphosphorylation.

Amino Acid Sequence↗
PubMed1997

The cardiac troponin C isoform and the length dependence of Ca2+ sensitivity of tension in myocardium.

The Ca2+ sensitivity of tension in cardiac muscle is length dependent, such that the sensitivity is diminished with decreasing sarcomere length below 2.4 microm. This length dependence of Ca2+ sensitivity of tension also forms the basis for the Frank-Starling mechanism in the heart. The fast-twitch skeletal muscle has a much lower length dependence of Ca2+ sensitivity. In a recent study of skinned cardiotrabeculae, we indicated that the exchange of endogenous cardiac troponin C (TnC) for skeletal troponin C also resulted in a major reduction in the length dependence to the level of skeletal muscle. These findings suggested that cardiac troponin C has a key role in the length-sensing mechanism. The present investigation supports this conclusion and delineates the specific domain in cardiac TnC responsible for the length effect. Chimeras splicing either 41, 61, or 96 N-terminal cardiac amino acids with the remaining skeletal residues have indicated that while Ca2+ binding in all three constructs is similar to that in wild type cardiac TnC, the functional responsiveness of the 96-cardiac residue construct is improved over the other two. This 96-cardiac residue construct yielded a tension response indistinguishable from that of wild-type cardiac TnC. A tryptophan variant of the chimera indicated fluorescence characteristics indistinguishable from cardiac troponin C. The findings provide further support for the idea that cardiac troponin C in situ is modified in response to sarcomere length change and thereby participates in the Frank-Starling mechanism. Moreover, the study indicates that the tropinin C length-sensing attribute originates within the N-terminal domain constituted by these 96 residues.

Animals↗
PubMed1996

Molecular basis of depression of Ca2+ sensitivity of tension by acid pH in cardiac muscles of the mouse and the rat.

BACKGROUND: Acid pH decreases the Ca2+ sensitivity of myocardial tension generation, and recent studies have suggested that regulatory proteins are involved. The current study defines the molecular basis of this effect on troponin C (TnC) and troponin I (TnI) and also addresses previous differences between the rat and mouse. METHODS AND RESULTS: Endogenous cardiac TnC and cardiac TnI in isolated trabeculae from mice and rats were exchanged with their fast-twitch skeletal muscle counterparts. A cardiac-skeletal TnC chimera was used to define the target region for proton action on cardiac TnC. Finally, cardiac TnC and skeletal TnC were genetically modified by insertion of a tryptophan for phenylalanine-26 to probe the pH effects with fluorescence spectroscopy. The pH 6.2 effects on Ca2+ sensitivity of force development in mouse and rat cardiotrabeculae are largely accounted for by the proton influences on TnC (23%) and TnI (53%). In cardiac TnC, residues 1 to 41 provide the target region. Comparison of the Ca(2+)-induced fluorescence in isolated cardiac TnC and skeletal TnC also indicated a greater pH effect in the cardiac isoform. CONCLUSIONS: The studies provide firm evidence that both TnC and TnI moieties are involved in the mechanism of acidosis causing reduction in the Ca sensitivity of force development in the myocardium. The findings rule out the possibility of interspecies variations in the underlying mechanisms. The genetically designed TnCs and a chimera demonstrate that the observed TnC-mediated difference in the pH effects on Ca2+ sensitivity of tension between cardiac and skeletal muscles is preserved in these isolated proteins. The N-terminal amino acid residues 1 to 41 in cardiac TnC are established as the pH sensor of this protein in the mouse as in the rat.

Animals↗
PubMed1995

Functional role of arginine-11 in the N-terminal helix of skeletal troponin C: combined mutagenesis and molecular dynamics investigation.

The two main structural differences between calmodulin (CaM) and skeletal troponin C (sTnC) are the absence in CaM of (i) the short N-terminal helix in TnC and (ii) the triplet KGK (residues 91-93; numbering according to chicken sTnC). It was recently shown that deletion of both structural groups from sTnC imparted to the resulting construct the CaM-like ability to activate phosphodiesterase (PDE) and to regulate force development in smooth muscle. To continue probing of the structural basis of the differential behavior of sTnC and CaM, residue Arg-11 in rabbit sTnC was mutated to Ala because the interactions of Arg-11 with distal residues in the N-terminal domain seem to link the N-terminal helix to the rest of the structure. The mutant exhibits CaM-like function in its ability to activate PDE (about 50% of CaM at 5 microM concentration). If, in addition, the KGK triplet is also deleted, PDE activation increases to about 80%. Both constructs retain their TnC function to nearly 100%. To explore the mechanistic basis of this remarkable observation, computational simulations of the molecular dynamics (MD) were carried out for both wild-type 4Ca2+.sTnC and the 4Ca2+.R11A mutant, and the results were compared to those from earlier simulations of 4Ca2+.CaM. Two types of structural changes observed from such simulations of the molecular dynamics of CaM had been considered to have a functional role: (i) a compaction to a more globular form and (ii) a reorientation of the Ca-binding domains around the central tether helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Contributions of troponin I and troponin C to the acidic pH-induced depression of contractile Ca2+ sensitivity in cardiotrabeculae.

Acid pH diminishes the Ca2+ sensitivity for force generation in both cardiac and skeletal muscles, but the mechanisms for these remain undetermined. In permeabilized (skinned) single myofibers of fast-twitch skeletal muscle of the rat, we find that pCa50 of the pCa-force relationship was 5.73 in pH 7 and 5.02 in pH 6.2 (delta pKskeletal = pCa50 in pH 7-pCa50 in pH 6.2 = 0.71 pCa unit); on the other hand, in skinned cardiotrabeculae, the hpCa50 was 5.79 in pH 7 decreasing to 4.14 in pH 6.2 (delta pKcardiac = 1.65 pCa units). We have used this large differential between cardiac/skeletal delta pKs to probe the mechanisms of the pH effects. Since troponin C (TnC) and troponin I (TnI) each have a central role in the Ca2+ switch, we exchanged these proteins in cardiac muscle with their skeletal counterparts and reinvestigated the pH effects. Firstly, with fast-twitch skeletal muscle (sTnC) substituting for 80% of the endogenous cardiac TnC (cTnC), the cardiac pH effect was decreased marginally (modified delta pK = 1.39 pCa units). This TnC-mediated change was further probed with two distinct cardiac-skeletal TnC chimeras, c1/s and CBc1/s (the Ca(2+)-binding c1/s), in which a majority of the N-terminal 41 amino acid residues was made cardiac and the rest skeletal [Gulati, J., & Rao, V. G. (1994) Biochemistry 33, 9052-9056]. The phenotype shift following sTnC/cTnC exchange in the trabeculae was blocked when c1/s was used in lieu of sTnC; on the other hand, interestingly, CBc1/s exactly mimicked sTnC.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1995

Molecular mobility of the Ca(2+)-deficient EF-hand of cardiac troponin C as revealed by fluorescence polarization of genetically inserted tryptophan.

To probe attitudinal features of the Ca(2+)-deficient site (site I) in the Ca2+ switch of cardiac troponin C (cTnC), we have examined steady-state fluorescence emission and polarization of a Trp26 inserted in a recombinant cardiac TnC (cTnC3.W) and compared these with the properties of the Ca(2+)-competent site I in skeletal TnC (sTnC4.W). The Ca(2+)-induced fluorescence emission in cTnC3.W was a fraction (25-30%) of that in sTnC4.W, in agreement with previous observations on the Ca(2+)-deficient site incorporated in a cardiac/skeletal chimera c1/s.W [Gulati, J. & Rao, V. G. (1994) Biochemistry 33, 9052-9056]. Thus, the fractional quantum yield reflected intrinsic properties of the cardiac metal ion-deficient site I. Conversely, in sTnC-1.W, where the skeletal site I also was made Ca(2+)-deficient by D27-->A substitution, the Ca(2+)-induced quantum yield was lower than that in cTnC3.W. Nevertheless, similar steady-state fluorescence polarizations for Ca(2+)-saturated sTnC4.W and cTnC3.W indicated indistinguishable final conformations in the two activated TnC isoforms. In EGTA, the polarization parameter (PEGTA) of sTnC4.W is greater than that of cardiac TnC, and the cardiac PEGTA value is closer to the activated PCa. Comparison of the chimera c1/s.W with sTnC-1.W indicated that the differences in conformation of the site I Trp for the EGTA-treated cardiac/skeletal isoforms were due to the structural disparities in this region. This contention was further supported by examination of the chimera CBc1/s.W, where the cardiac EF-hand was altered by 27VLGA30-->DAD substitution. Polarization of the relaxed form was similar to that for sTnC4.W. These findings suggest that the relaxed conformation of the cardiac Ca2+ switch is more favorably predisposed to activation than the skeletal switch.

Animals↗
PubMed1995

Diminished Ca2+ sensitivity of skinned cardiac muscle contractility coincident with troponin T-band shifts in the diabetic rat.

We have measured the apparent Ca2+ sensitivities of force development in skinned cardiac trabeculae at different sarcome lengths together with shifts in troponin (Tn) T subunits on specimens from the same hearts and drawn insights into the pathogenesis of myocardial dysfunction in the diabetic rat. The Ca(2+)-force relations were measured at a long (2.4-microns) and a short (1.9-microns) sarcomere length. In disease, compared with the control condition, the apparent Ca2+ sensitivity was greatly diminished at a sarcomere length of 1.9 microns but not affected at all at the long length (2.4 microns). We also examined the alterations in contractile regulatory proteins TnT and TnI by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots. The TnI band was largely unperturbed, but major changes were discerned in TnT. The normal rat heart indicated two major bands (TnT1 and TnT2) and a faint third band (TnT3); in the diabetic rat heart, there was a significant shift in intensity from TnT1 to TnT3. Since myosin isozyme shifts also accompany diabetes in the rat, we used a prototypical hypothyroid rat as well to evaluate the myosin influence in the length-induced effects on Ca2+ sensitivity. Myosin shifts during hypothyroidism were unaccompanied by significant changes in TnT, and there were also no length-dependent modifications in Ca2+ sensitivity. The findings raise the possibility that diabetic Ca(2+)-sensitivity changes in the myocardium are coupled with TnT alterations. A plausible explanation is offered whereby these TnT alterations modify the length dependence of Ca2+ sensitivity.

Animals↗
PubMed1994

Disparate contributions of Tyr10 and Tyr109 to fluorescence intensity of rabbit skeletal muscle troponin C identified using a genetically engineered mutant.

Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca(2+)-induced conformational changes in troponin C (TnC). Rabbit skeletal TnC contains two tyrosines (Y10 in the N-helix, and Y109 in site 3 in the C-terminal domain) in distinct microenvironments: their individual contributions to total fluorescence intensity are elucidated here utilizing bacterially synthesized rabbit skeletal TnC (sTnC4) and a genetically engineered variant, termed 109YF, lacking one of the tyrosines (Y109 replaced with F109). The steady-state fluorescence emission spectra following excitation at 280 nm were recorded in EGTA (Ca(2+)-free) and Ca(2+)-saturated (pCa4) solutions. For the wild-type sTnC4, pCa4 causes a significant (46%) increase in the peak fluorescence intensity over the value in EGTA. For the mutant 109YF, the EGTA fluorescence is only marginally affected (74% of the wild-type FEGTA), but interestingly the Ca2+ effect is completely suppressed (delta F = FpCa4-FEGTA = 2% of the wild-type value). These results indicate that the two tyrosines make disparate contributions to the fluorescence spectrum of wild-type sTnC, both in the presence and absence of Ca2+; whereas Y10 in the N-helix is dominant in Ca(2+)-free solution, Y109 is the sole contributor to the Ca2+ effect. Furthermore, to explain the biphasic fluorescence response of Y109 obtained during Ca2+ titrations, the findings yield the most unequivocal evidence that Ca(2+)-induced conformational changes in the trigger sites operating the contractile switch modify properties of the C-terminal sites in TnC pari passu.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

Shifts in contractile regulatory protein subunits troponin T and troponin I in cardiac hypertrophy.

To examine the molecular basis of hypertrophied heart failure, we investigated the changes in cardiac contractile regulatory proteins. The guinea pigs were subjected to chronic pressure overload with aortic banding to induce ventricular hypertrophy, and in-situ pressure-volume relations were recorded together with biochemical characterizations to ascertain the contractile modifications. Immunoblots of left and right ventricular samples revealed four distinct troponin T isoforms, which underwent alterations during hypertrophy. The higher molecular weight bands TnT1 and TnT2 shifted towards the lower molecular weight isoforms TnT3 and TnT4. For TnI, a single prominent band was detected, whose intensity also increased with pump failure. The findings provide the first direct evidence of TnT and TnI shifts in an experimentally induced hypertrophied heart failure and has novel mechanistic implications for the future studies.

Animals↗
PubMed1994

The role of glycine (residue 89) in the central helix of EF-hand protein troponin-C exposed following amino-terminal alpha-helix deletion.

Because an N-terminal alpha-helical (N-helix) arm and a KGK-triplet (residues 88KGK90) in the central helix of troponin-C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structure-function correlations of these units. Presently, with a family of genetically manipulated derivatives especially developed for this study and tested on permeabilized isolated single skeletal muscle fiber segments, we explored the specificities of the amino acid residues within the N-helix and the KGK-triplet in TnC. Noticeably, the amino acid compositions vary between the N-helices of the cardiac and skeletal TnC isoforms. On the other hand, the KGK-triplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N-helix (mutant delta Nt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK90-triplet (mutant delta Nt delta KGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268:11685-11690). Using this functional test, we find that replacement of Gly-89 with a Leu or an Ala could also overcome the contractile defect associated with N-helix deletion. On the other hand, replacement of the skeletal TnC N-helix with cardiac type N-helix was unable to restore contractile function. The findings indicate a destabilizing influence of Gly-89 residue in skeletal TnC and suggest that the N-terminal arm in normal TnC serves to moderate this effect. Moreover, specificity of the N-helix between cardiac and skeletal TnCs raises the possibility that resultant structural disparities are also important for the functional distinctions of the TnC isoforms.

Amino Acid Sequence↗
PubMed1993

Critical minimum length of the central helix in troponin C for the Ca2+ switch in muscular contraction.

In the troponin C (TnC) dumbbell, the NH2- and COOH-terminal lobes are well delineated, but the role of the central helix and especially the function of its long length remain doubtful. To study this, we used a cDNA construct encoding rabbit fast-twitch muscle TnC, comprising multiple restriction sequences to facilitate mutagenesis (Babu, A., Su, H., Ryu, Y. & Gulati, J. (1992) J. Biol. Chem. 267, 15469-15474). Systematically, we have deleted 3-12 amino acid residues from the central helix and examined their effects in maximally activated skinned muscle fibers. Limiting the deletions to 7 amino acid residues manifested little change in maximal force development (Sheng, Z., Francois, J. M., Hitchcock, S. E. & Potter, J. D. (1991) J. Biol. Chem. 266, 5711-5715). However, with further deletions, we now find that contractility was inhibited pari passu; by 12 deletions, the inhibition was complete. The critical minimum length for the central helix is thereby estimated as 27 A. The Ca2+ binding capacity (4 mol of Ca2+/mol of protein) as well as the structural characteristics (alpha-helicity by CD measurements and the fluorescence emitted by Tyr-109) indicated a well preserved global conformation of the short mutant. However, surprisingly, two of these short mutants filled each TnC slot under highly specific superloading conditions: one short molecule was taken up in EGTA solution, and the second molecule was captured and retained with Ca2+. They also rescued the contractile switch, evidently in a bimolecular reaction. Another short variant (putative skeletal fast muscle TnC-I-II), in which the NH2-terminal Ca(2+)-binding sites were incapacitated, failed to respond to superloading, indicating that sites III and IV could not substitute for sites I and II. The results suggest that a critical role of the central helix linker in TnC is to keep the two lobes optimally apart, evidently in proximity of their respective target sites on troponin I in the fiber.

Amino Acid Sequence↗
PubMed1993

Identification of the regions conferring calmodulin-like properties to troponin C.

The structural and functional correlations between troponin C (TnC) and calmodulin (CaM) were investigated by mutagenizing a synthetic cDNA coding rabbit skeletal muscle TnC. Compared with TnC, calmodulin lacks the N-terminal alpha-helical arm (N-helix), and its central helix is shorter due to the absence of 88KGK90 residues. Deleting both regions concomitantly (delta Nt delta KGK) elicited CaM-like regulation as tested (i) by smooth muscle contractility (maximal tension = 80 +/- 5% Po of control) and (ii) by the activation of phosphodiesterase (Vmax = 75 +/- 2% of control). The Ca(2+)-binding capacity of the mutant and the effect of the mutant on maximally Ca(2+)-activated tension of skinned rabbit psoas muscle fibers were both conserved. Furthermore, in the linker region of the central helix, replacing the TnC-characteristic 85EDAKGK90 successive residues with CaM-specific DTD residues generated a highly effective CaM mimic (Vmax = 96 +/- 2%) whether or not the N-helix was also retained. Apparent KD values (i.e. concentrations for half-maximal response) for the successful mutants were similar to each other but about 200-fold higher than that for CaM. A part of the alpha-helical linker region in CaM may unfold and bend to promote multiplicity of target interaction using all four hands (Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638; Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) Science 257, 1251-1255). In contrast, our results suggest that the TnC central helix evolved to be less pliable by the combined influences of 85EDAKGK90 residues and the alpha-helical extension in N terminus, thereby keeping the N-terminal hands well separated from their C-terminal counterparts.

Amino Acid Sequence↗
PubMed1993

The mechanism of Ca(2+)-coordination in the EF-hand of TnC, by cassette mutagenesis.

Genetic engineering of TnC and skinned fiber physiology on rabbit psoas muscle are combined to study the mechanisms of Ca(2+)-binding in the EF-hand in TnC. Of the six coordinating positions (X,Y,Z,-Y,-X & -Z) for Ca(2+)-binding in the loop, the X position is invariably occupied by an aspartate, and the -Z position by a glutamate. X-ray analysis has indicated that both oxygen atoms of the beta-carboxylate in aspartate (in X) are extensively hydrogen bonded to other residues in the loop. When this aspartate in site II was replaced by a glutamate (gamma-carboxylate), Ca(2+)-binding was annihilated, and the mutant was unable to regulate force development in the fiber. Similarly, glutamate for aspartate exchange in the -Z position of site I also inactivated the site as well as its function in skinned fiber. Mutations in the Y position indicated that a glutamate was unacceptable in place of aspartate but that an asparagine was acceptable. The Ca(2+)-sensitivity with asparagine was also similar to that of the wild type. The study indicates a powerful approach for defining the physicochemical principles governing Ca-coordination and sensitivity in Ca-binding proteins. Furthermore, by comparison with findings on chemically synthesized peptides, the results show that behavior of the EF-hand in TnC is modified by quaternary structure of the molecule.

Amino Acid Sequence↗
PubMed1993

Molecular biology of the length-tension relation in cardiac muscle.

TnC in the thin filament is found to act as the putative length-sensor in cardiac muscle. Techniques of genetic engineering and TnC-exchange in skinned fibers are combined to delineate the molecular principles governing the length induced modulations in TnC. Cardiac TnC is a better length-sensor than skeletal TnC, and the differences in the two moieties are used as a guide to explore the length-sensing mechanism. Studies are described with mutants of both cardiac and skeletal TnCs: (i) a mutant of cardiac TnC in which Ca(2+)-binding to site 1 was restored (made by John Putkey), and (ii) two mutants of skeletal TnC, (a) with site 1 inactivated, and (b) a chimera, in which 41 residues in the N-terminus are entirely of the cardiac type and the remaining residues are all of the skeletal type (made at Albert Einstein). The results indicate that Ca(2+)-binding property of site 1 is only partly responsible for the length-sensitivity of TnC, and that the residues outside loop-1 are also critical in both the on-off switching mechanism as well as the length-sensing mechanism. Further, we have also found that thin filaments are polarized such that Ca(2+)-affinity may be high at the free end of the filament and decreases towards the Z-line, reaching its lowest value at the fixed end. This could serve as the basis for the conversion of sarcomere length change into a signal that can be transduced from the Z-lines to the TnC moiety in actin filaments.

Amino Acid Sequence↗
PubMed1992

Functional delineation of the Ca(2+)-deficient EF-hand in cardiac muscle, with genetically engineered cardiac-skeletal chimeric troponin C.

Cardiac and fast skeletal isoforms of TnC each comprise four putative EF-hand (helix-loop-helix) motifs as potential Ca(2+)-binding sites (sites 1-4), except that site 1 in cardiac TnC is deficient in Ca2+ coordination. In skeletal TnC, the N-terminal sites 1 and 2 are both essential for the trigger mechanism of the contraction switch. However, the mechanism in cardiac muscle is unsettled; it is obscure whether the cardiac site 1 is functionally inert due to calcium deficiency and consequently site 2 is the lone trigger site, or whether sites 1 and 2 perform interactively despite the impairment. These possibilities were addressed by mutagenizing site 1 in skeletal TnC to mimic the cardiac response. In one mutant (STnC-1), two selected Ca(2+)-ligands were abolished. In another (C1/S chimera), 41 N-terminal residues from cardiac TnC were spliced to STnC. The Ca(2+)-binding capacities as well as skinned fiber responses were measured. The STnC-1 derivative failed to switch on contraction. In contrast, the chimeric construct expressed close to full contractile potential in myocardium (74 +/- 3% Po; Po = maximal tension) and also the manifest cardiac phenotype. By devising supplemental chimeric constructs, cardiac-type N-terminal overhang together with cardiac-type EF-hand for site 1 both were found essential for the phenotype. We conclude that cardiac TnC site 1 is actively engaged in the trigger mechanism and in fact dominates the phenotype despite the inability to chelate Ca2+. The N-terminal overhang also participates in this mechanism, which is a novel finding. The conclusion that a non-chelating site functions interactively with a proximal site in cardiac TnC may have wider significance, inasmuch as similar pairings of disparate EF-hands are of common occurrence.

Amino Acid Sequence↗