CONFORMATIONAL CHANGES OF ACTIN INDUCED BY STRONG OR WEAK MYOSIN SUBFRAGMENT-1
BINDING
I. V. Dedova,1 S. V. Avrova,2 N. N. Vikhoreva2
P. G. Vikhorev2
T. L. Hazlett,3 W. Van der Meer,4 C. G. Dos Remedies,1
Yu. S. Borovikov2 *
1 Institute of Biomedical Research, University of Sydney, Australia,
2 Institute of Cytology RAS, St. Petersburg, Russia,
3 Department of Physics and Astronomy, Western Kentucky University, USA, and
4 Laboratory for Fluorescence Dynamics, University of Illinois, USA;
* e-mail: boroviko@mail.cytspb.rssi.ru
Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S 1 binding were
studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes
attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373
labeled with NBD-C1; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with
1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in
filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes).
The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore
location and binding site of actomyosin. Strong binding of S1 with actin leads to 1) a decrease in the orientation of oscillators of derivatives
of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in
the "front" surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right
and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a
weak binding of SI to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis
cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin
conformation play an important role in muscle contraction.
Key words: G-actin; F-actin; actin-myosin interaction; conformational changes of actin; intramolecular and intermolecular
movements; myosin subfragment-1; ghost muscle fibers; fluorescence polarization
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