Interactions of Structural C and Regulatory N Domains of Troponin C with Repeated Sequence Motifs in Troponin I

Abstract

The actomyosin ATPase inhibitory protein troponin I (TnI) plays a central regulatory role in skeletal and cardiac muscle contraction and relaxation through its calcium-dependent interactions with troponin C (TnC) and actin. Previously we have demonstrated the utility of F29W and F105W mutants of TnC for measurement of binding affinities of inhibitory peptide TnI(96-116) to its regulatory N and structural C domains, both in isolation and in the intact TnC molecule [Pearlstone, J. R. & Smillie, L. B. (1995) Biochemistry 34, 6932-6940]. This approach is now extended to fragment TnI(96-148). Curve-fitting analyses of fluorescence changes induced in the intact TnC mutants and the isolated N and C domains by increasing [TnI(96-148)] have permitted the assignments of K(D) values (designated K(D,N) and K(D,C)) to the interaction of TnI(96-148) with the N and C domains, respectively, of intact TnC. Taken together with the previous data for TnI(96-116) binding, it can be concluded that, within TnI(96-148), residues 96-116 are primarily responsible for binding to C domain of intact TnC and residues 117-148 to its N domain. Inspection of the available mammalian and avian skeletal muscle TnI amino acid sequences reveals a previously unrecognized conserved motif repeated 3-fold, once in the inhibitory peptide region (approximately residues 101-114; designated alpha) and twice more in the region of residues approximately 121-132 (beta) and approximately 135-146 (gamma). The number and distribution of these motifs have important structural implications for the TnI x C complex. In the beta motif of cardiac TnI, as compared with skeletal, several changes in charged amino acids are suggested as candidates responsible for the greater sensitivity of cardiac Ca2+-regulated actomyosin to acidic pH as in ischemia.

Mutations of Hydrophobic Residues in the N-terminal Domain of Troponin C Affect Calcium Binding and Exchange with the Troponin C-Troponin I96–148 Complex and Muscle Force Production

“…1) on Ca 2ϩ binding and exchange in the presence of the regulatory peptide of TnI, TnI 96 -148 . The N-terminal domain of TnC F29W undergoes a large increase in Trp fluorescence upon forming the Ca 2ϩ -TnC F29W -TnI 96 -148 complex (8,20). In the absence of Ca 2ϩ , the Trp fluorescence of TnC F29W upon the addition of TnI 96 -148 marginally decreased Ͻ1.1-fold at 345 nm ( Fig.…”

“…Therefore, substitution of hydrophobic residues with polar Gln produced N-domain TnC F29W mutants that exhibited ϳ243-fold variation in their Ca 2ϩ affinities in the presence of TnI 96 -148 . The Hill coefficients for all but two of the TnC F29W -TnI 96 -148 mutant complexes (F26QTnC F29W and I37QTnC F29W ) were between 1.6 and 2.8 (see Table I (20). Hydrophobic interactions are important for the Ca 2ϩ -dependent binding of the N-domain of TnC to the C-domain of TnI.…”

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

“…Together with TnI, TnC is one of the three subunits of muscle troponin complex and a protein naturally binds TnI with high affinity (7). Meantime, TnC is a eukaryotic protein that can readily express at very high levels in E. coli.…”

“…Therefore, the ineffective binding between TnI and TnC in bacterial cells might not be simply due to the solution environment but result from ineffective folding of the bacterially made troponin proteins (most likely cardiac TnI since the TnC protein was in excess and only a small portion needed to be correctly folded to saturate the co-expressed cardiac TnI). It has been demonstrated by us and many other laboratories that TnI and TnC purified from bacterial expression both have biochemical activities and can form functional TnI-TnC binary complex and tertiary troponin complex [3,7,10]. These results indicate that denaturing (urea buffer) and renaturing (final dialysis) steps during the purification of bacterial made TnI are necessary for the yield of biologically active proteins.…”

“…In the present study, we investigated the factors that limit the expression of cardiac TnI in E. coli. To reduce the potential toxicity of cardiac TnI to the host cell, we constructed a bi-cistronic expression vector to co-express cardiac TnI and cardiac/slow TnC, a natural binding partner of TnI and a protein that readily expresses in E. coli at very high levels [7]. To examine the effect of prokaryotic usage of eukaryotic codons on the translational efficiency of cardiac TnI mRNA, we compared expression in a host E. coli strain containing additional tRNAs for certain low bacterial usage eukaryotic codons with that in host bacteria containing only E. coli tRNAs.…”