Lys-553 of skeletal muscle myosin subfragment 1 (S1) was specifically label
ed with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexa
noic acid succinimidyl ester) and fluorescence quenching experiments were c
arried 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 q
uenchers of varying charge [nitromethane, (2,2,6,6-tetramethyl-1-piperinylo
xy) (TEMPO), iodide (l(-)), and, thallium (Tl+)] were used to assess both t
he 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 tha
llium, 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 app
ears to interact differently with actin in the weakly and strongly bound st
ates.