Bc. Phan et al., EFFECT OF COMPLEXES OF ADP AND PHOSPHATE ANALOGS ON THE CONFORMATION OF THE CYS707-CYS697 REGION OF MYOSIN SUBFRAGMENT-1, European journal of biochemistry, 243(3), 1997, pp. 636-642
Recent crystallographic studies have suggested structural differences
between the complexes of S1 . Mg . ADP with the phosphate analogs alum
inium fluoride (AlF4-), vanadate (VO43-) and beryllium fluoride (BeFx)
[Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Ho
lden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972; Smith, R.
& Rayment, I. (1996) Biochemistry 35, 5404-5417]. In this work, chemi
cal modifications, namely labeling of Cys707 (the reactive SH1 thiol)
and Cys707-Cys697 (SH1-SH2) cross-linking, were used to compare the S1
. ADP . BeFx, S1 . ADP . AlF4- and S1 . ADP . VO43- complexes with sp
ecific states of the myosin-ATPase pathway. Modification of Cys707 wit
h the fluorescent monofunctional reagents thylamino-3-(4'-maleimidylph
enyl)-4-methylcoumarin and N-iodoacetyl-N'-(5-sulfo-1-naphtyl)ethyl ha
s shown that the reactivity of the SH1 group depends on the nucleotide
bound to S1. The observed rates of Cys707 modification at 20 degrees
C lead to the conclusion that S1 . ADP . BeFx is similar to S1 . ATP,
while S1 . ADP . AlF4- and S1 . ADP . VO43- are more similar to S1*
. ADP . P-i. The conformations of the analog states were also compared
by monitoring the dissociation of the fluorescent nucleotide analog 1
-N-6-ethenoadenosine diphosphate (ADP[C2H2]) from the active site of C
ys707-modified (by N-ethylmaleimide) and Cys707-Cys697-cross-linked (b
y N,N'-p-phenylene dimaleimide) S1 . ADP[C2H2] . AlF4- and S1 . ADP[C2
H2] . BeFx. Our results suggest that the conformations of the S1 . ADP
. AlF4-, S1 . ADP . VO43- and S1 . ADP . BeFx complexes in the Cys707
-Cys697 region are distinct from each other, with the former two at le
ast partially resembling the S1* . ADP . P-i state, while the latter
is similar to the prehydrolyzed S1 . ATP state.