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 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 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 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 approximately 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.
Leblanc
L
L
Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Bennet
A
A
Borgford
T
T
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D003956:Dialysis; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019209:Troponin C; D002118:Calcium