Distance between Cys-374 of Actin and Lys-553 of Myosin
in a Rigor Complex Determined by Fluorescence Resonance Energy Transfer
C.M. Yengo* and C.L. Berger
*Department of Molecular Physiology & Biophysics and Department
of Biochemistry, University of Vermont, Burlington, VT 05405-0068.
We have used fluorescence resonance energy transfer to determine the
distance between IAEDANS at Cys-374 on actin and fluorescein at Lys-553
of myosin subfragment 1 (S1) in order to help elucidate the molecular interactions
responsible for the formation of the rigor acto-S1 complex. Cys-374 of
actin was selectively modified with 1,5-IAEDANS (((((2-iodoacetyl) amino)
ethyl)amino) naphthalene-1-sulfonic acid) by the method of Kasprzak et
al. (1988, Biochemistry 27: 4512). Lys-553 of S1 was selectively
labeled with FHS (6-(fluorescein-5-carboxamido)hexanoic acid, succinimidyl
ester) by the method of Bertrand et al. (1995, Biochemistry 34:9500).
R0 for the IAEDANS-actin - FHS-S1 donor-acceptor
pair was determined to be 44.03 Å, assuming an orientation factor
(
2) was 2/3. Fluorescence
energy transfer efficiencies were determined by comparing the IAEDANS actin
lifetime in the absence of bound FHS-S1 (
D
= 18.03 1.02 ns) and in the presence of saturating FHS-S1 to form a 1:1
rigor complex between IAEDANS-actin and FHS-S1 (DA
= 3.05 ± 1.40 ns). The calculated fluorescence energy
transfer efficiency of 83% corresponds to a distance (r) of 33.8 Åbetween
IAEDANS on Cys-374 of actin and Lys-553 of myosin S1. Our observed value
of r is larger than that predicted by modeling the atomic structures of
actin and S1 within 3-D reconstructions of the rigor acto-S1 complex (>>22
Å; Rayment et al. (1993) Science 261:58). Although
this may be due in part to the size and flexibility of the fluorescent
probes used in this work, our results impose important experimentally-derived
constraints on the way in which actin and myosin can interact with each
other in the rigor complex.
This work was supported by grants to C.L.B. from the American Heart
Association (960145000), the New Hampshire-Vermont Affiliate of the American
Heart Association (9606223S), and the National Institutes of Health (AR
44219-01).
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