Spatial relationship between the nucleotide‐binding site, Lys‐61 and Cys‐374 in actin and a conformational change induced by myosin subfragment‐1 binding

Abstract

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 EATP or formycin-A 5'-triphosphate (FTP) and Cys-374 was labelled with 5-{ 2-[(iodoacetyl)amino]ethyl}aminonaphthalene-l-sulfonic acid (1,SIAEDANS) 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 k 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,SIAEDANS 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, 229-2351, 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,SIAEDANS attached to Cys-374 in its nearest-neighbour monomer in an F-actin filament was calculated to be 3.34 k 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-I (Sl) 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 a 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.Actin is widely present in all eukaryotic cells and possesses several functions in cellular movement and cytoskeletal structure. The polymerization of monomeric actin (G-actin) to a double-helical structure (F-actin) is an essential function of actin.According to the model derived from X-ray crystallography of the actin-DNase-I complex [l], actin consists of two domains of slightly different size, with the small domain containing the N-terminus. The phosphate moiety of the bound nucleotide is located in the cleft and DNase I binds to a loop region in the small domain where residues 50 -69 lie since these residues are chemically cross-linked to DNase I selectively labelled with chemical reagents in G-actin but not in F-actin. The chemical modification of these residues impairs the polymerization of actin. Moreover, the bonds between Lys-61 and Arg-62 and between Lys-68 and T...

Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein

“…2-4). The binding was accompanied by marked changes in the fluorescence emission of IAEDANS attached to Cys 374 in subdomain 1 at the barbed face of G-actin (44), as reported previously to other WH2/ Thymosin␤4 proteins( Fig. 2A) (12,45).…”

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

“…Fluorescence labeling was carried out in the same buffer. Cys 374 of actin was labeled with the donor (IAEDANS) following polymerization as described earlier (28).…”

Actin as the generator of tension during muscle contraction.

“…Third, the zigzagging appearance and overall width of r-actin closely resemble the wide view of h-actin seen by electron microscopy (8), while the narrowness of the edge-on view (9) and fiber diffraction data (10) seriously limit the orientational possibilities for the monomer in the helical fiber. Fourth, fluorescence energy measurements enabled the locations of nucleotide and cysteines to be triangulated with respect to the filament axis, establishing the monomer orientation (11).…”