Frequency-domain lifetime fluorometry of double-labeled creatine kinase.
Myofibril-bound creatine kinase EC 2.7.3.2 (CK), a key enzyme of muscle energy metabolism, has been selected for studies of conformational changes that underlie the cellular control of enzyme activity. For fluorescence spectroscopy measurements, the CK molecule was double-labeled with IAF (5-iodoacetamidofluorescein) and ErITC (erythrosin 5'-isothiocyanate). Measurement of fluorescence resonance energy transfer (FRET) from fluorescein to erythrosin was used to obtain information about the donor-acceptor pair distance. Frequency-domain lifetime measurements evaluate the donor-acceptor distance in the native CK molecule as 7.8 nm. The Förster radius equals 5.3 nm with the resolution range from 0.2 to 1.0 nm. Erythrosin-fluorescein labeling (EFL) was tested for artificial conformational changes of the CK molecule with high-salt concentration treatment. The transition distance, defined by His-97 and Cys-283 and derived from a 3D model equals 0.766 nm for the open (inactive) form and 0.277 nm for the closed (reactive) form of the CK molecule. In this way, the resolution range of the used spectroscopy method is significant, concerning the difference of 0.489 nm. Nevertheless, the CK enzyme activity, assessed by the hexokinase-coupled assay, was diminished down to 1 % of the activity of the native enzyme. EFL is suitable for description of conformational behavior implied from the regulation of creatine kinase. However, the observed inhibition restricts EFL to studies of conformational changes during natural catalytic activity.
Gregor
M
M
Department of Physiology and Developmental Biology, Faculty of Sciences, Charles University, Vinicná 7, CZ-128 44 Prague 2, Czech Republic. gregor@natur.cuni.cz
Kubala
M
M
Amler
E
E
Mejsnar
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004923:Erythrosine / Q000031:analogs & derivatives* / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D017879:Isothiocyanates / Q000737:chemistry; D009994:Osmolar Concentration; D010725:Phosphocreatine / Q000378:metabolism; D011189:Potassium Chloride / Q000737:chemistry; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry
D005452:Fluoresceins; D017879:Isothiocyanates; D013438:Sulfhydryl Compounds; D010725:Phosphocreatine; C015296:5-iodoacetamidofluorescein; D011189:Potassium Chloride; C022232:erythrosine isothiocyanate; D000255:Adenosine Triphosphate; D003402:Creatine Kinase; D003545:Cysteine; D004923:Erythrosine