Nanosecond study of fluorescently labeled troponin C.
The time-resolved extrinsic fluorescence of rabbit skeletal troponin C was studied with the protein labeled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Both the intensity and anisotropy decays followed a biexponential decay law, regardless of the ionic condition, pH, viscosity or temperature. The lifetimes and their fractional amplitudes were insensitive to Mg2+, and the lifetimes were also insensitive to Ca2+. In response to Ca2+ binding to all four sites, the fractional amplitude (alpha 1) associated with the short lifetime (tau 1) decreased by a factor of two, thus increasing the ratio of the two amplitudes alpha 2/alpha 1 from 1.6 to 4.3. These amplitude changes suggest the existence of two conformational states of TnC-IAEDANS, with the conformation associated with the long-decay component (tau 2) being promoted by saturation of the two Ca(2+)-specific sites. At pH 5.2 the ratio alpha 2/alpha 1 for the apo-protein was 3.5 indicating different relative populations of the two decay components when compared with pH 7.2. In the presence of Ca2+ at the lower pH, alpha 2/alpha 1 decreased to 2.1, suggesting a shift of the conformations in favor of the short-decay component. Thus Ca2+ elicited different conformational changes in TnC at the two pH values. The recovered anisotropies suggest that there were fast molecular motions that were not resolved in the present experiments, and some of these motions were sensitive to Ca2+ binding to the specific sites. These results support the notion of communication between the N-domain and the C-terminal end of the central helix of troponin C.
Wang
C K
CK
Department of Biochemistry, University of Alabama, Birmingham 35294.
Liao
R
R
Cheung
H C
HC
D000818:Animals; D005456:Fluorescent Dyes; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009132:Muscles / Q000378:metabolism; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014336:Troponin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019209:Troponin C
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D014336:Troponin; D019209:Troponin C; C006421:1,5-I-AEDANS