Spatial relationship between the nucleotide-binding site, Lys-61 and Cys-374 in actin and a conformational change induced by myosin subfragment-1 binding.
The spatial relationship between Lys-61, the nucleotide binding site and Cys-374 was studied. Lys-61 was labelled with fluorescein-5-isothiocyanate as a resonance energy acceptor, the nucleotide-binding site was labelled with the fluorescent ATP analogues epsilon ATP or formycin-A 5'-triphosphate (FTP) and Cys-374 was labelled with 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) as a resonance energy donor. The distances between the nucleotide binding site and Lys-61 or between Lys-61 and Cys-374 were calculated to be 3.5 +/- 0.3 nm and 4.60 +/- 0.03 nm, respectively. (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) On the other hand, when doubly-labelled actin with 1,5-IAEDANS at Cys-374 and FITC at Lys-61 was polymerized in the presence of a twofold molar excess of phalloidin [Miki, M. (1987) Eur. J. Biochem. 164, 229-235], the fluorescence of 1,5-IAEDANS bound to actin was quenched significantly. This could be attributed to inter-monomer energy transfer. The inter-monomer distance between FITC attached to Lys-61 in a monomer and 1,5-IAEDANS attached to Cys-374 in its nearest-neighbour monomer in an F-actin filament was calculated to be 3.34 +/- 0.06 nm, assuming that the likely change in the intra-monomer distance does not change during polymerization by more than 0.4 nm. One possible spatial relationship between Lys-61, Cys-374 and the nucleotide binding site in an F-actin filament is proposed. The effect of myosin subfragment-1 (S1) binding on the energy transfer efficiency was studied. The fluorescence intensity of AEDANS-FITC-actin decreased by 30% upon interaction with S1. The fluorescence intensity of AEDANS-FITC-actin polymer in the presence of phalloidin increased by 21% upon interaction with S1. The addition of ATP led to the fluorescence intensity returning to the initial level. Assuming that the change of fluorescence intensity can be attributed to conformational change in the actin molecule induced by S1 binding, the intra-monomer distance was reduced by 0.4 nm and the inter-monomer distance was increased by 0.2 nm.
D000199:Actins / Q000032:analysis*; D000345:Affinity Labels; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis*; D004735:Energy Transfer; D008239:Lysine / Q000032:analysis*; D015879:Myosin Subfragments; D009218:Myosins / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D010590:Phalloidine / Q000494:pharmacology; D011108:Polymers / Q000032:analysis; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000199:Actins; D000345:Affinity Labels; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011108:Polymers; D010590:Phalloidine; D009218:Myosins; D008239:Lysine; D003545:Cysteine