Cm. Yengo et al., Intrinsic tryptophan fluorescence identifies specific conformational changes at the actomyosin interface upon actin binding and ADP release, BIOCHEM, 38(44), 1999, pp. 14515-14523
The helix-loop-helix (A-site) and myopathy loop (R-site) are located on opp
osite sides of the cleft that separates the proposed actin-binding interfac
e of myosin. To investigate the structural features of the A- and R-sites,
we engineered two mutants of the smooth muscle myosin motor domain with the
essential light chain (MDE), containing a single tryptophan located either
in the A-site (W546-MDE) or in the R-site (V413W MDE). W546- and V413W-MDE
display actin-activated ATPase and actin-binding properties similar to tho
se of wild-type MDE. The steady-state fluorescence properties of W546-MDE [
emission peak (lambda(max)) = 344, quantum yield = 0.20, and acrylamide bim
olecular quenching constant (k(q)) = 6.4 M-1 . ns(-1)] and V413W-MDE [lambd
a(max) = 338, quantum yield = 0.27, and k(q) = 3.6 M-1 . ns(-1)] demonstrat
e that Trp-546 and Trp-413 are nearly fully exposed to solvent, in agreemen
t with the crystallographic data on these residues. In the presence of acti
n, Trp-546 shifts to a more buried environment in both the ADP-bound and nu
cleotide-free (rigor) actomyosin complexes, as indicated by an average lamb
da(max) of 337 or 336 nm, respectively, and protection from dimethyl(2-hydr
oxy-5-nitrobenzyl) sulfonium bromide (DHNBS) oxidation. In contrast, Trp-41
3 has a single conformation with an average lambda(max) of 338 nm in the AD
P-bound complex, but in the rigor complex it is 50% more accessible to DHNB
S oxidation and can adopt a range of possible conformations (lambda(max) =
341-347 nm). Our results suggest a structural model in which the A-site rem
ains tightly bound to actin and the R-site adopts a more flexible and solve
nt-exposed conformation upon ADP release.