Photoaffinity labeling of skeletal myosin with 2-azidoadenosine triphosphate.
The purine binding site of ATP on skeletal muscle myosin has been photoaffinity labeled with 2-azidoadenosine diphosphate (2-N3ADP). 2-N3ADP was stably trapped at the active site (t1/2 approximately 5 days, 0 degree C) by complexation of the two heavy chain reactive thiols (Cys-697 and Cys-707) with Co(III)phenanthroline. Photoincorporation occurred only in the 23-kDa NH2-terminal tryptic fragment of the heavy chain. Extensive serial digestion of photolabeled subfragment 1 of myosin by trypsin and subtilisin yielded a series of labeled peptides which were purified by HPLC. Sequence and radiolabeling analysis of eight photolabeled peptides all indicated that tryptophan-130 was the only labeled residue. This site of labeling confirms earlier photolabeling studies with the non-nucleotide ADP analogue, 2[(4-azido-2-nitrophenyl)-amino]ethyl diphosphate (NANDP), which also labeled Trp-130 [Okamoto, Y., & Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1579]. Comparison of the structures of 2-N3ADP and NANDP indicate that their azido groups can be superimposed if both analogues bind to the active site in an extended conformation in a manner analogous to the anti conformation of ATP.
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
J C
JC
Grammer
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Kuwayama
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RG
Yount
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry; D011817:Rabbits; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D015879:Myosin Subfragments; D010446:Peptide Fragments; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D009218:Myosins