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

Site-directed mutagenesis was used to make amino acid substitutions at posi t-ion 54 of skeletal troponin C, testing a relationship between the stabili ty 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 bindi ng site II. Mutations were made in the context of a previously described ph enylalanine 29-->tryptophan (F29W) variant (Trigo-Gonzalez ef at, Biochemis try 31, 7009-7015 (1992), which allows binding events to be monitored throu gh changes in the intrinsic fluorescence of the protein. N-Cap substitution s 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 x 10(-6), 1.36 x 10(-6), 2.09 x 10(-6), 2.28 x 10(-6), and 4.24 x 10(-6) M, respectively. The steady state binding of calcium to proteins in the mu tant series was seen to be monophasic and cooperative. Calcium off-rates we re measured by stopped how fluorescence and in every instance two transitio ns were observed. The rate constant of the first transition, corresponding to similar to 99% of the change in fluorescence, was between 900 +/- 20 and 1470 +/- 100 s(-1), whereas the rate constant of the second transitions wa s between 94 +/- 9 and 130 +/- 23 s(-1). The significance of two transition s remains unclear, though both rate constants occur on a time scale consist ent with the regulation of contraction, (C) 2000 Academic Press.