title: Time-resolved fluorescence study of the single tryptophans of engineered skeletal muscle troponin C.
abstract: The regulatory domain of troponin C (TnC) from chicken skeletal muscle was studied using genetically generated mutants which contained a single tryptophan at positions 22, 52, and 90. The quantum yields of Trp-22 are 0.33 and 0.25 in the presence of Mg2+ (2-Mg state) and Ca2+ (4-Ca state), respectively. The large quantum yield of the 2-Mg state is due to a relatively small nonradiative decay rate and consistent with the emission peak at 331 nm. The intensity decay of this state is monoexponential with a single lifetime of 5.65 ns, independent of wavelength. In the 4-Ca state, the decay is biexponential with the mean of the two lifetimes increasing from 4.54 to 4.92 ns across the emission band. The decay-associated spectrum of the short lifetime is red-shifted by 19 nm relative to the steady-state spectrum. The decay of Trp-52 is biexponential in the 2-Mg state and triexponential in the 4-Ca state. The decay of Trp-90 requires three exponential terms for a satisfactory fit, but can be fitted with two exponential terms in the 4-Ca state. The lower quantum yields (< 0.15) of these two tryptophans are due to a combination of smaller radiative and larger nonradiative decay rates. The results from Trp-22 suggest a homogeneous ground-state indole ring in the absence of bound Ca2+ at the regulatory sites and a ground-state heterogeneity induced by activator Ca2+. The Ca(2+)-induced environmental changes of Trp-52 and Trp-90 deviate from those predicted by a modeled structure of the 4-Ca state. The anisotropy decays of all three tryptophans show two rotational correlation times. The long correlation times (phi 1 = 8.1-8.3 ns) derived from Trp-22 and Trp-90 suggest an asymmetric hydrodynamic shape. TnC becomes more asymmetric upon binding activator Ca2+ (phi 1 = 10.1-11.6 ns). The values of phi 1 obtained from Trp-52 are 3-4 ns shorter than those from Trp-22 and Trp-90, and these reduced correlation times may be related to the mobility of the residue and/or local segmental flexibility.
authors.affiliation: Department of Physics, University of Alabama at Birmingham 35294, USA.
authors.forename: M
authors.initials: M
authors.lastname: She
authors.forename: W J
authors.initials: WJ
authors.lastname: Dong
authors.forename: P K
authors.initials: PK
authors.lastname: Umeda
authors.forename: H C
authors.initials: HC
authors.lastname: Cheung
mesh_terms: D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002645:Chickens; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D005454:Fluorescence Polarization / Q000379:methods; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects; D011789:Quantum Theory; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D019209:Troponin C / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects; D014364:Tryptophan
chemical_list: D000178:Acrylamides; D011994:Recombinant Proteins; D019209:Troponin C; D020106:Acrylamide; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium