Key Points
- To determine the structural location of the ATPase catalytic site on the myosin head using photoaffinity labeling and electron microscopy.
- Synthesized a biotinylated photoreactive ADP analog and trapped it at a 1:1 molar ratio inside the ATPase site of heavy meromyosin (HMM) or myosin subfragment 1 (S1) using vanadate ion.
- Crosslinked the analog to the protein using ultraviolet illumination and mapped the crosslinked segment on the heavy chain.
- Bound an avidin oligomer marker to the biotin moiety and visualized the complex using rotary-shadowed electron microscopy.
- Approximately 15% of the trapped ADP analog covalently crosslinked to the protein upon ultraviolet illumination, mapping specifically to the N-terminal 25,000-Da segment of the heavy chain.
- Rotary-shadowed electron microscopy images of the HMM-avidin complex revealed that the ATPase site is located approximately 140 Å from the head-rod junction along the myosin head.
Structured PICO
PPopulationHeavy meromyosin (HMM) or myosin subfragment 1 (S1)
IInterventionPhotoaffinity labeling with a biotinylated photoreactive ADP analog and UV illumination
OOutcomeVisualization of the ATPase site of myosin and mapping of the crosslinking site
This basic science study mapped the ATPase site of myosin to be approximately 140 A from the head-rod junction using electron microscopy and photoaffinity labeling.