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ATP induces microsecond rotational motions of myosin heads crosslinked to actin
Abstract:
We have used saturation transfer electron paramagnetic resonance (ST-EPR) to study the effect of ATP on the rotational dynamics of spin-labeled myosin heads crosslinked to actin (XLAS1). We have previously shown that ATP induces microsecond rotational motions in activated myofibrils or muscle fibers, but the possibility remained that the motion occurred only in the detached phase of the cross-bridge cycle. The addition of ATP to the crosslinked preparation has been shown to be a model system for active cross-bridges, presumably providing an opportunity to measure the motion in the attached state, without interference from unattached heads. In the absence of ATP, XLAS1 had very little microsecond rotational mobility, yielding a spectrum identical to that observed for uncrosslinked acto-S1. This suggests that all of the labeled S1 forms normal rigor complexes when crosslinked to actin. The addition of 5 mM ATP greatly increased the microsecond rotational mobility of XLAS1, and the effects were reversed upon depletion of ATP. The most plausible explanation for these results is that myosin heads undergo microsecond rotational motion while attached actively to actin during steady state ATPase activity. These results have important implications for the interpretation of spectroscopic data obtained during muscle contraction.
Insights
Adenosine triphosphate (ATP) drives microsecond rotational motion in myosin heads attached to actin. This finding, observed using saturation transfer electron paramagnetic resonance (ST-EPR), clarifies cross-bridge dynamics during muscle contraction.
Area of Science:
- Biophysics
- Muscle Physiology
- Biochemistry
Background:
- Muscle contraction involves the dynamic interaction of actin and myosin.
- Previous studies indicated ATP-induced motion in myofibrils, but its occurrence in the attached state was uncertain.
- The crosslinked acto-S1 (XLAS1) preparation serves as a model for attached cross-bridges.
Purpose of the Study:
- To investigate the effect of ATP on the rotational dynamics of spin-labeled myosin heads attached to actin.
- To determine if microsecond rotational motions occur in the attached phase of the cross-bridge cycle.
Main Methods:
- Saturation Transfer Electron Paramagnetic Resonance (ST-EPR) spectroscopy was employed.
- Spin-labeled myosin heads (S1) were crosslinked to actin (XLAS1).
- The rotational mobility of XLAS1 was measured in the presence and absence of ATP.
Main Results:
- In the absence of ATP, XLAS1 exhibited minimal microsecond rotational mobility, similar to uncrosslinked acto-S1.
- Addition of 5 mM ATP significantly increased the microsecond rotational mobility of XLAS1.
- ATP-induced mobility was reversible upon ATP depletion.
Conclusions:
- Myosin heads undergo microsecond rotational motion while actively attached to actin during steady-state ATPase activity.
- These findings are crucial for interpreting spectroscopic data from contracting muscle.
- The study provides evidence for dynamic conformational changes in attached cross-bridges.