Abstract
The heavy chain of myosin subfragment-1 (S1) is cleaved by limited trypsinolysis into three fragments, 27, 50, and 20 kDa--aligned in this order from the N-terminus. The tertiary structure of the molecule is essentially not affected by trypsinolysis. The spatial relations between the various regions of the molecule and the nucleotide- and actin-induced intramolecular movements were studied by cross-linking tryptic S1 with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), phenylenediglyoxal (PDG), and glutaraldehyde. The formation of cross-linked products was monitored by SDS-PAGE, using the fluorescent probes 9-anthronitrile and N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS), which specifically label the 27- and 20-kDa fragments, respectively. The reaction with the cross-linkers leads to the formation of 50-kDa/20-kDa, 27-kDa/20-kDa, 27-kDa/50-kDa, and 20-kDa/light chain cross-linked products. Of these, the most intensive was the formation of the 50-kDa/20-kDa products, which appeared as a doublet on the SDS-PAGE with all the cross-linkers. This indicates that the interface between the two fragments is rather extended. The presence of MgATP or MgADP promoted the formation of the 20-kDa/50-kDa cross-linked products, especially with the lower electrophoretic mobility band, when EEDQ was used as a cross-linker. With PDG as a cross-linker, MgATP also affected the cross-link formation between the 20-kDa fragment and the light chains whereas it had no influence on the formation of other products. On the other hand, the effect of actin on the cross-linking with the various cross-linkers was quite extensive, and it was manifested in the reduction of cross-link formation between the various S1 domains. It is concluded that both nucleotides and actin induce intramolecular movements in S1 and that the nucleotide-induced movements are more restricted than those induced by actin, which extend to larger regions of the molecule.
Published Version
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