Proximity and ligand-induced movement of interdomain residues in myosin subfragment 1 containing trapped MgADP and MgPPi probed by multifunctional cross-linking.
The conformations of myosin subfragment 1 containing trapped MgADP or MgPPi have been studied by investigating the spatial disposition of the remainder of the subfragment 1 structure to the covalently bridged ATPase-related thiols SH1 and SH2. This has been done by synthesizing a trifunctional photoactivatable reagent 4,4'-bis(N-maleimido)benzophenone and reacting it with subfragment 1 in the presence of these ligands. Modification of subfragment 1 by this reagent mimics closely the changes in the ATPase properties as noted previously for modification with p-phenylenedimaleimide. In addition, noncovalent trapping of nucleotide also results, presumably by the bridging of the SH1 and SH2 thiols. On photolysis, cross-linking from the reagent bridging the thiols to other regions in subfragment 1 can be observed, but the extent and course of the photoinduced cross-linking depend on the nature of the trapped ligand. For subfragment 1 with trapped MgADP, a high efficiency cross-linking occurs between the 21-kDa segment and the 50-kDa segment. With MgPPi as the trapped ligand, low efficiency cross-linking occurs between the bridged thiols and either the 27-kDa N-terminal or the 50-kDa segments of the heavy chain. These results indicate that without the adenosine moiety, the binding of MgPPi to subfragment 1 leaves the protein in a flexible state so that residues in both the 27-kDa and the 50-kDa segment can move within the cross-linking span of the activated benzophenone triplet. The trapping of MgADP apparently results in a more rigid state for the subfragment 1 in which residues in the 50-kDa segment are spatially close to the bridged thiols, thus enabling photocross-linking to proceed with higher efficiency.
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D001577:Benzophenones; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D011756:Diphosphates / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008274:Magnesium / Q000378:metabolism*; D017616:Magnesium Compounds; D008297:Male; D008970:Molecular Weight; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010782:Photolysis; D011487:Protein Conformation; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014357:Trypsin
D001577:Benzophenones; D003432:Cross-Linking Reagents; D011756:Diphosphates; D017616:Magnesium Compounds; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D000244:Adenosine Diphosphate; C053364:4,4'-bis(N-maleimido)benzophenone; C034732:magnesium pyrophosphate; D014357:Trypsin; D009218:Myosins; D008274:Magnesium