Calcium affinity of regulatory sites in skeletal troponin-C is attenuated by N-cap mutations of helix C.
Substance Nomenclature: | 0 (Troponin C) SY7Q814VUP (Calcium) |
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Entry Date(s): | Date Created: 20010523 Date Completed: 20010614 Latest Revision: 20131121 |
Update Code: | 20231215 |
DOI: | 10.1006/abbi.2000.2103 |
PMID: | 11368316 |
Autor: | Leblanc L; Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada., Bennet A, Borgford T |
Jazyk: | angličtina |
Zdroj: | Archives of biochemistry and biophysics [Arch Biochem Biophys] 2000 Dec 15; Vol. 384 (2), pp. 296-304. |
DOI: | 10.1006/abbi.2000.2103 |
Abstrakt: | 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. |
Databáze: | MEDLINE |
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