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Biophys J, March 2000, p. 1441-1448, Vol. 78, No. 3
Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine, Burlington, Vermont 05405-0068 USA
Lys-553 of skeletal muscle myosin subfragment 1 (S1) was
specifically labeled with the fluorescent probe FHS
(6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl
ester) and fluorescence quenching experiments were carried out to
determine the accessibility of this probe at Lys-553 in both the
strongly and weakly actin-bound states of the MgATPase cycle. Solvent
quenchers of varying charge [nitromethane,
(2,2,6,6-tetramethyl-1-piperinyloxy) (TEMPO), iodide (I
),
and thallium (Tl+)] were used to assess both the steric
and electrostatic accessibilities of the FHS probe at Lys-553. In the
strongly bound rigor (nucleotide-free) and MgADP states, actin offered
no protection from solvent quenching of FHS by nitromethane, TEMPO, or
thallium, but did decrease the Stern-Volmer constant by almost a factor
of two when iodide was used as the quencher. The protection from iodide
quenching was almost fully reversed with the addition of 150 mM KCl,
suggesting this effect is ionic in nature rather than steric.
Conversely, actin offered no protection from iodide quenching at low
ionic strength during steady-state ATP hydrolysis, even with a
significant fraction of the myosin heads bound to actin. Thus, the
lower 50 kD subdomain of myosin containing Lys-553 appears to interact differently with actin in the weakly and strongly bound states.
Biophys J, March 2000, p. 1441-1448, Vol. 78, No. 3
© 2000 by the Biophysical Society 0006-3495/00/03/1441/08 $2.00
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