Calcium affinity of regulatory sites in skeletal troponin-C is attenuated by N-cap mutations of helix C.

Abstract Site-directed mutagenesis was used to make amino acid substitutions at position 54 of skeletal troponin C, testing a relationship between the stability of helix C and calcium ion affinity at regulatory sites in the protein. Normally, threonine at position 54 is the first helical residue, or N-cap, of the C helix; where helices C and D, and the loop between, comprise binding site II. Mutations were made in the context of a previously described phenylalanine 29→tryptophan (F29W) variant (Trigo-Gonzalez et al., Biochemistry 31, 7009–7015 (1992)), which allows binding events to be monitored through changes in the intrinsic fluorescence of the protein. N-Cap substitutions at position 54 were shown to attenuate the calcium affinity of regulatory sites in the N-terminal domain. Calcium affinities diminished according to the series T54 T54S > T54A > T54V > T54G with dissociation constants of 1.36 × 10 −6 , 1.36 × 10 −6 , 2.09 × 10 −6 , 2.28 × 10 −6 , and 4.24 × 10 −6 M, respectively. The steady state binding of calcium to proteins in the mutant series was seen to be monophasic and cooperative. Calcium off-rates were measured by stopped flow fluorescence and in every instance two transitions were observed. The rate constant of the first transition, corresponding to ∼99% of the change in fluorescence, was between 900 ± 20 and 1470 ± 100 s −1 , whereas the rate constant of the second transitions was between 94 ± 9 and 130 ± 23 s −1 . The significance of two transitions remains unclear, though both rate constants occur on a time scale consistent with the regulation of contraction.