Fluorescence quenching studies of fluorescein attached to Lys-61 or Cys-374 in actin: effects of polymerization, myosin subfragment-1 binding, and tropomyosin-troponin binding.
The resonance energy transfer between fluorescein-5-isothiocyanate (FITC) attached to Lys-61 and Co2+ bound to the high-affinity metal binding site was measured. The distance between FITC and Co2+ on the actin molecule was calculated to be either 1.9 nm, using the absorption spectrum of Co-EDTA or 2.8 nm, using the absorption spectrum of Co2+ bound to carboxypeptidase as a model spectrum of Co2+ bound to actin, respectively. The effects of the polymerization of actin and of the interaction of actin with myosin subfragment-1 (S1) on the solvent accessibility of the fluorescein molecule attached to Lys-61 or Cys-374 were measured. The accessibility of the probe at Lys-61 was reduced following polymerization and also appreciably reduced by interaction with S1. The accessibility of the probe attached to Cys-374 was affected to only a small degree. These results indicate that the Lys-61 residue is located close to an actin-actin contact region as well as being close to an S1 binding site, although it is not directly involved [Miki, M. (1987) Eur. J. Biochem. 164, 228-235]. The accessibility of the probe at Lys-61 was also decreased by the addition of the tropomyosintroponin complex, although the accessibility of the probe at Cys-374 was not affected at all. Thus, Lys-61 appears to be involved in the binding site of the regulatory proteins.
D000199:Actins / Q000032:analysis*; D003545:Cysteine / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005453:Fluorescence; D008239:Lysine / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011108:Polymers; D011485:Protein Binding; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates; D014335:Tropomyosin / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism*; D014783:Viscosity
D000199:Actins; D005452:Fluoresceins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011108:Polymers; D012997:Solvents; D013861:Thiocyanates; D014335:Tropomyosin; D014336:Troponin; D009218:Myosins; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine; D003545:Cysteine