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Dynamics at Lys-553 of the acto-myosin interface in the weakly and strongly bound states.

Biophysical journal (2000-02-29)
J J MacLean, L R Chrin, C L Berger
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
6-[Fluorescein-5(6)-carboxamido]hexanoic acid, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
6-[Fluorescein-5(6)-carboxamido]hexanoic acid N-hydroxysuccinimide ester, suitable for fluorescence, ≥75% (HPLC)

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