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An interdomain distance in cardiac troponin c determined by fluorescence spectroscopy
Author(s) -
Dong WenJi,
Robinson John M.,
Xing Jun,
Cheung Herbert C.,
Umeda Patrick K.
Publication year - 2000
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.9.2.280
Subject(s) - troponin complex , troponin i , chemistry , biophysics , troponin c , binding site , tryptophan , fluorescence , acceptor , crystallography , biochemistry , physics , amino acid , biology , optics , psychology , psychiatry , myocardial infarction , condensed matter physics
The distance between Ca 2+ ‐binding site III in the C‐terminal domain and Cys35 in the N‐terminal domain in cardiac muscle troponin C (cTnC) was determined with a single‐tryptophan mutant using bound Tb 3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb 3+ was generated through sensitization by the tryptophan located in the 12‐residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48–49 Å regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half‐width >9 Å) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 Å for cTnC, but without an effect on the half‐width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.