Three-dimensional image analysis of myosin head in function as captured by quick-freeze deep-etch replica electron...

Area of Science:

• Biophysics
• Structural Biology
• Molecular Motors

Background:

• Understanding the dynamic structural changes of myosin crossbridges is crucial for elucidating muscle contraction mechanisms.
• Existing techniques often lack the resolution or preservation needed to visualize individual, functioning protein molecules in solution.

Purpose of the Study:

• To investigate the three-dimensional structure and conformational changes of heavy meromyosin (HMM) crossbridges during muscle activity.
• To visualize individual functioning myosin molecules under in vitro motility assay conditions.

Main Methods:

• Quick-freeze deep-etch replica electron microscopy combined with the mica-flake technique.
• Three-dimensional reconstruction of single HMM molecules using filtered back-projection from tilted images.

Main Results:

• Under rigor conditions, both HMM heads are straight and bind actin at a 45-degree angle.
• During in vitro sliding, HMM attaches to actin via a single head at various angles.
• Free HMM heads exhibit a kinked conformation with ATP or ADP/vanadate, contrasting with their straight form without nucleotide.
• 3-D reconstruction revealed structural differences in HMM-ADP/Vi compared to the nucleotide-free atomic model, suggesting conformational changes.

Conclusions:

• The study provides high-resolution structural insights into individual myosin crossbridges during function.
• Nucleotide binding significantly alters HMM head conformation and crossbridge attachment dynamics.
• The findings contribute to a deeper understanding of the molecular mechanisms underlying muscle contraction.