Changes in Molecular Motions of the Essential Light Chain
Accompany Structural Changes Induced by Phosphorylation of Regulatory Light
Chain
Bishow Adhikari, Joshua Somerset and Piotr G. Fajer
Institute of Molecular Biophysics., Florida State University, National
High Magnetic Field Laboratory, Tallahassee FL 32306
The relative geometry of the catalytic domain of myosin head with respect
to the filament backbone is a function of pH, ionic strength (µ),
and the regulatory light chain (RLC) phosphorylation (Levine et al., 1995;
Ludescher et al., 1988). Since the mobility of the catalytic domain is
decoupled from that of regulatory domain (Adhikari et al., 1995), we now
examine whether the two domains behave in the same way. ELC was labeled
with InVSL at the cysteine-176 and exchanged into myosin. ST-EPR was used
to determine protein mobility. Increasing µ or pH both resulted in
an increase of myosin head mobility. At pH 7.0, the effective rotational
correlation times (
R) at
µ = 45, 125, and 200 mM were 18-26, 14-22, and 12-13 ms, respectively.
At pH 6.5, 7.0 and 8.2 the mobility was respectively, R
= 19-24, 18-26 and 6-10 ms. These trends follow those reported for the
catalytic domain (Ludescher et al., 1988). Thus the dynamic response of
the head to the above modulators encompasses both domains. The RLC was
phosphorylated enzymatically using MLCK (gift of Dr J. Stull) and CaM.
2D gels showed that most of the RLC was phosphorylated under these conditions.
Phosphorylated myosin filaments, R=
16 ± 2 ms, were more mobile than the dephosphorylated myosin, R=
25 ± 2 ms, under identical conditions. Therefore, the displacement
of the phosphorylated heads observed by EM (Levine et al., 1995) is accompanied
by the increased mobility.
The Effect of Myosin Head State on the Orientation of
Troponin C
Hui-chun Li and Piotr G. Fajer
Department of of Biological Science, Florida State University, National
High Magnetic Field Laboratory, Tallahassee FL 32306
In vertebrate skeletal muscle, contraction is initiated by elevation
of intracellular Ca2+ concentration. The steric blocking model
suggests that Ca2+ binding to the N-terminal of troponin C subunit,
results in conformational changes in the thin filament proteins that expose
the myosin-binding site on actin. Besides Ca2+, myosin head
also plays a role in the activation of thin filaments (Lehrer, 1994). Our
previous study has shown that troponin C reorients upon binding of calcium
and/or myosin heads attachment, and the orientational distribution of TnC
is different in response to Ca2+ and in response to rigor heads
(Li & Fajer, 1994). This work examines TnC reorientation induced by
different chemical state of myosin head bound to actin filaments in muscle
fibers. As before, we have used electron paramagnetic resonance of spin
labeled TnC reconstituted into muscle fibers. Five biochemical intermediate
states of myosin were trapped by chemical perturbation: (a) M*ATP, detached
heads; (b) A*M*ATP, weakly bound heads (at low ionic strength and low temperature);
(c) A*M*ADP*Pi, strongly bound pre-power stroke state; (d) A*M*ADP, strongly
bound post-power stroke state; and (e) A*M, strongly bound rigor state.
The orientational distribution of TnC is the same in weakly bound states
and detached myosin heads. TnC reorients significantly when myosin heads
enter strongly bound states. The orientation of TnC in pre-power stroke
state is different from that in the post-power stroke state. This findings
imply a multitude of conformational states of TnC and their coupling to
the actomyosin states in agreement with the model of Geeves and Lehrer.
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