Abstract:
:Fluorescence lifetimes of dimeric rabbit muscle creatine kinase specifically dansylated at both active sites and the homologous monomeric lobster muscle arginine kinase singly dansylated were determined using phase-modulation methods with global analysis of overdetermined data sets. For both proteins, the data is adequately described by three discrete exponential decays or a Lorentzian double distributed decay. Analogue phase resolved spectroscopy also reveals the presence of at least two distinct fluorophore domains for the dansyl moieties of creatine kinase. The model fluorophore, dansyllysine, exhibits a monoexponential decay with a value that is highly solvent dependent. Because the monomeric arginine kinase exhibits essentially the same decay law as doubly derivatized dimeric creatine kinase, it is proposed that the multiple lifetimes of creatine kinase reflect two or more isomeric dimeric states and not subunit asymmetry within a conformationally homogeneous dimeric population. Exposure of arginine kinase to 6 M guanidinium chloride results in a shift to shorter lifetimes and narrowing of the lifetime distributions. Creatine kinase displays a small narrowing of the distribution, but little change in fractional populations or lifetimes. These results suggest the presence of structural elements resistant to denaturation. The longest lifetime component in the triexponential discrete decay law of doubly dansylated creatine kinase is totally unquenched by acrylamide, whereas the two shorter lifetime components exhibit limited dynamic quenching. Steady-state quenching by acrylamide is significant and reveals a sharp distinction between accessible and non accessible dansyl groups. The major mechanism for interaction between the dansyl moieties and acrylamide is, atypically, static quenching. The results are consistent with two dansyl domains, one accessible and hydrophilic according to lifetime values and the other inaccessible and hydrophobic in solvent characteristics.Energy transfer between the dansyl group and the eight tryptophan residues of dimeric creatine kinase give similar results(~ 35%) from measurements of lifetimes, steady-state donor quenching and sensitized acceptor emission. The similarity suggests that the overall flexibility of the dimeric protein is limited. The occurrence of multiple conformers of muscle creatine kinase provides an explanation for several previous observations, most notably the structural origins for compartmentation of the muscle isozyme observed in the myofibril.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Grossman SHdoi
10.1016/S0006-3495(91)82274-3subject
Has Abstractpub_date
1991-03-01 00:00:00pages
590-7issue
3eissn
0006-3495issn
1542-0086pii
S0006-3495(91)82274-3journal_volume
59pub_type
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