Cm. Yengo et al., Interaction of myosin LYS-553 with the C-terminus and DNase I-binding loopof actin examined by fluorescence resonance energy transfer, J STRUCT B, 131(3), 2000, pp. 187-196
Fluorescence resonance energy transfer (FRET) experiments were carried out
in the absence of nucleotide (rigor) or in the presence of MgADP between fl
uorescent donor probes (IAEDANS (5((((2-iodoacetyl)amino)ethyl)amino)-napht
halene-1-sulfonic acid) at Cys-374 or DANSYL (5-dimethylamino naphthalene-1
-(N-(5-aminopentyl))sulfonamide) at Gln-41 of actin and acceptor molecules
(FHS (6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester
) at Lys-553 of skeletal muscle myosin subfragment 1. The critical Forster
distance (R-0) was determined to be 44 and 38 Angstrom for the IAEDANS-FHS
and DANSYL-FHS donor-acceptor pairs, respectively. The efficiency of energy
transfer between the acceptor molecules at Lys-553 of myosin and donor pro
bes at Cys-374 or Gln-41 of actin was calculated to be 0.78 +/- 0.01 or 0.9
4 +/- 0.01, respectively, corresponding to distances of 35.6 +/- 0.4 Angstr
om and 24.0 +/- 1.6 Angstrom respectively. MgADP had no significant effect
on the distances observed in rigor. Thus, rearrangements in the acto-myosin
interface are likely to occur elsewhere than in the lower 50-kDa subdomain
of myosin as its affinity for actin is weakened by MgADP binding. (C) 2000
Academic Press.