Introduction of a donor-acceptor pair by a single protein modification. Förster energy transfer distance measurements from trapped 1,N6-ethenoadenosine diphosphate to chromophoric cross-linking reagents on the critical thiols of myosin subfragment.
The fluorescent ADP analog, 1,N6-ethenoadenosine diphosphate (epsilon ADP), has been trapped at the active site of myosin subfragment 1 (SF1) by the chromophoric cross - linkers, 1,5 - difluoro - 2,4' - dinitrobenzene (F2DNB) and 4,4'-difluoro-3,3'-dinitrophenylsulfone (F2DPS). The cross-linking agents were shown to react with the kinetically reactive cysteines SH1 and SH2 (i) by virtue of their effect on the characteristic K+-EDTA and CaATPase activities of SF1, (ii) by the loss of two thiols after reaction of enzyme with equimolar concentrations of cross-linking agent, and (iii) by comparison of the uv absorption spectra of cross-linked SF1 with those of the thiol adducts of F2DPS and F2DNB. In addition, F2DPS was shown to be located predominantly on the 20 kDa heavy chain tryptic peptide fragment known to contain SH1 and SH2. The fluorescence decay of the epsilon ADP-SF1 complex was found to be heterogenous by phase modulation methods after reaction with the cross-linking reagents F2DPS and F2DNB. The resolved lifetimes were found to be 26.1 and 7.0 ns for the F2DPS system and 25.2 and 3.1 ns for the F2DNB system, indicating the presence of some free, as well as trapped and quenched, nucleotide in both systems. The shorter lifetimes (Förster energy transfer quenched) and the spectral overlap for the two systems were used to calculate distances of 26 A and 23 A between the purine binding site and the enzyme adducts of F2DPS and F2DNB, respectively. These distance measurements demonstrate that both SH1 and SH2 are too far from the active site to be directly involved in either the binding or the hydrolysis of ATP.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives / Q000494:pharmacology; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010446:Peptide Fragments; C013936:difluorodinitrobenzene sulfone; C015195:1,N(6)-ethenoadenosine diphosphate; D000244:Adenosine Diphosphate; D004139:Dinitrofluorobenzene; D000251:Adenosine Triphosphatases; D009218:Myosins; C002041:1,5-difluoro-2,4-dinitrobenzene