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Single-headed binding of a spin-labeled-HMM-ADP complex to F-actin. Saturation transfer electron paramagnetic
Abstract:
The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl-imidodiphosphate (AMPPNP) to determine the contributions of single and double-headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results.
Insights
This study investigated actin and spin-labeled heavy meromyosin (MSL-HMM) interactions. Results show adenosine diphosphate (ADP) promotes single-headed binding, while AMPPNP does not, clarifying myosin head binding mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Myosin motor proteins interact with actin filaments to generate force and movement.
- Understanding the binding modes of myosin heads (single- vs. double-headed) is crucial for elucidating muscle contraction mechanisms.
Purpose of the Study:
- To differentiate between single- and double-headed binding of spin-labeled heavy meromyosin (MSL-HMM) to actin.
- To investigate the influence of nucleotides like adenosine diphosphate (ADP) and AMPPNP on these binding modes.
Main Methods:
- Utilized saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the fraction of immobilized spin-labeled myosin heads (fi).
- Employed sedimentation assays to determine the fraction of bound myosin molecules (fs).
- Analyzed changes in fi and fs upon nucleotide titration of acto-MSL-HMM complexes.
Main Results:
- Adenosine diphosphate (ADP) titration showed minimal change in fs but a significant decrease in fi, indicating extensive single-headed binding.
- AMPPNP titration resulted in parallel decreases in both fi and fs, suggesting no significant single-headed binding was detected.
- The observed binding behaviors were explained without invoking nucleotide-induced conformational changes in bound heads.
Conclusions:
- Distinguishes the binding mechanisms of ADP and AMPPNP with actin-bound heavy meromyosin.
- Provides evidence for prevalent single-headed binding of MSL-HMM to actin in the presence of ADP.
- Demonstrates that AMPPNP binding does not favor single-headed interactions, simplifying the understanding of myosin-actin dynamics.