Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog.
An ADP analog carrying a biotin moiety and a photoreactive group was synthesized. In the presence of vanadate ion (Vi), the analog was tightly trapped into the ATPase site of heavy meromyosin (HMM) or myosin subfragment 1 (S1) in an ADP analog/ATPase site molar ratio of 1:1. UV illumination on the HMM (or S1)-Vi-ADP analog complex resulted in covalent incorporation of the analog into the ATPase site. About 15% of the trapped analog was crosslinked to HMM or S1. Mapping of the crosslinking site of the analog showed that the N-terminal Mr 25,000 segment of the heavy chain participated in binding the ADP analog. The biotin moiety of the analog covalently incorporated into the ATPase site was visualized in electron microscopy by attaching an avidin oligomer. Rotary-shadowed images of the HMM-avidin complex revealed that the crosslinked ADP analog was located about 140 A from the head-rod junction on the head. The result indicates that the ATPase site of myosin is about 140 A apart from the head-rod junction along the head.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D000251:Adenosine Triphosphatases; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001710:Biotin; D008854:Microscopy, Electron / Q000379:methods; D008970:Molecular Weight; D015879:Myosin Subfragments; D009218:Myosins; D010446:Peptide Fragments; D010777:Photochemistry; D011817:Rabbits
D000345:Affinity Labels; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D001710:Biotin; D000251:Adenosine Triphosphatases; D009218:Myosins