Inhibition of mutant troponin C activity by an intra-domain disulphide bond

Abstract

Triggering of contraction in striated muscles involves a conformational transition in the N-terminal domain of troponin C, the calcium-binding component of thin filaments. We have designed a mutant troponin C in which the key conformational transition and the calcium-regulatory activity are reversibly blocked by the formation of a disulphide bridge. Our results may be applicable to other proteins of the same family of calcium-binding proteins.

Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C

“…This value is in a range of previously reported Ca 2ϩ affinities for bovine cTnC F27W at 7°C (10.7 M) and 21°C (5.1 M) (27). Residues Phe 20 and Met 81 are located within the NAD unit, whereas Val 44 , Met 45 , and Leu 48 are located within the BC unit. All of these residues are almost completely buried in the absence and presence of Ca 2ϩ (with an exception of Leu 48) (Table I and Ref. 15), with their side chains involved in extensive hydrophobic interactions between the BC and NAD units (Fig.…”

“…However, the N-domain of cTnC remains essentially closed in the Ca 2ϩ bound state, because the BC unit moves away only slightly from the NAD unit (15). Therefore, analogous residues in cTnC, Phe 20 , Val 44 , Met 45 , Leu 48 , and Met 81 exhibit no increase in their solvent accessibility upon Ca 2ϩ binding (Table I) and form numerous side chain contacts with each other in both the apo and Ca 2ϩ -bound states. Thus, there was a question of whether analogous mutations in cTnC would have the same effect on Ca 2ϩ affinity and exchange rates as they did in sTnC.…”

“…No correlation was found between the Ca 2ϩ affinity of the 27 mutants and the solvent accessibility of the mutated residues in either the absence or presence of Ca 2ϩ (or the difference between the two states), indicating that the changes in solvent exposure were not the sole determinant in how the residue affects Ca 2ϩ binding. Substitution of Phe 22 , Val 45 Met 46 , Leu 49 , or Met 82 with polar Gln led to large (3.2-18.9-fold) increases in the Ca 2ϩ affinity. We concluded that these mutations increased Ca 2ϩ affinity because they shift the equilibrium from the apo state toward the Ca 2ϩ -bound state by reducing the hydrophobic contact between the BC and NAD units in the apo state.…”

Cloning and expression of chicken skeletal muscle troponin I in Escherichia coli: The role of rare codons on the expression level

“…The fact that the three troponin subunits can refold and reassociate in vitro (Greaser & Gergely, 1971) has allowed the use of troponin subunits produced in Escherichia coli to study the molecular mechanism of this regulatory complex. Expression of TnC in bacteria (Chen et al, 1988;Reinach & Karlsson, 1988;Xu & HitchcockDeGregori, 1988) and the analysis of site-directed mutants (Fujimori et al, 1990;Grabarek et al, 1990;Putkey et al, 1991;Sheng et a]., 1991;Negele et al, 1992;Pearlstone et al, 1992;Silva et al, 1993) in conjunction with the determination of the crystal structure of TnC (Herzberg & James, 1985;Sundarlingam et al, 1985) has provided a better understanding of the calcium-induced conformational change in TnC (Silva & Reinach, 1991;Grabarek et al, 1992). This conformational change is responsible for the modulation of the inhibitory action of Tnl.…”

John Gergely (1919–2013): a pillar in the muscle protein field

“…The calcium and magnesium binding properties of TnC and the troponin complex Gergely 1974, 1975) were next established. Subsequent work using a multitude of spectroscopic (Leavis et al 1978;Wang et al 1982) and cross-linking (Leszyk et al 1990;Grabarek et al 1990;Luo et al 2000) techniques have provided the foundation for our current understanding of the protein structural changes in the calcium control of muscle contraction.…”