C-TERMINAL SITES OF CALDESMON DRIVE ATP HYDROLYSIS CYCLE BY SHIFTING
ACTOMYOSIN ITERMEDIATES FROM STRONG TO WEAK BINDING OF MYOSIN AND ACTIN
O. E. Pronina,1 O. Copeland,2 S. Marston,2
Yu. S. Borovikov 1, *
1 Institute of Cytology, RAS, St. Petersburg, Russia, and
2 National Heart and Lungs Institute, London, UK;
* e-mail: boroviko@mail.cytspb.rssi.ru
Polarized fluorimetry technique and ghost muscle fibers containing tropomyosin were used to study effects
of caldesmon (CaD) and recombinant peptides CaDH1 (residues 506—793), CaDH2 (residues 683—767),
CaDH12 (residues 506—708) and 658C (residues 658—793) on the orientation and mobility of fluorescent label
1.5—IAEDANS specifically bound to Cys-707 of myosin subfragment-1 (S1) in the absence of nucleotide,
and in the presence of MgADP, MgAMP-PNP, MgATPγS or MgATP. It was shown that at modelling different
intermediates of actomyosin ATPase, the orientation and mobility of dye dipoles changed discretely, suggesting
a multi-step changing of the myosin head structural state in ATP hydrolysis cycle. The maximum difference in
orientation and mobility of the oscillator (4° and 30 %, respectively) was observed between actomyosin in the
presence of MgATP, and actomyosin in the presence of MgADP. Caldesmon actin-binding sites C and BR inhibit
formation of actomyosin strong binding states, while site B activates it. It is suggested that actin-myosin interaction
in ATP hydrolysis cycle initiates nucleotide-dependent rotation of myosin motor domain, or that of its site
for dye binding as well as the change in myosin head mobility. Caldesmon drives ATP hydrolysis cycle by shifting
the equilibrium between strong and weak forms of actin-myosin binding.
Key words: regulation of muscle contraction, ATP analogues, intermediate states of actomyosin, caldesmon,
changes of myosin subfragment-1 conformation, fluorescence polarization
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