MOLECULAR-MOVEMENTS IN THE ACTOMYOSIN COMPLEX - F-ACTIN-PROMOTED INTERNAL CROSS-LINKING OF THE 25-KDA AND 20-KDA HEAVY-CHAIN FRAGMENTS OF SKELETAL MYOSIN SUBFRAGMENT-1

We describe, for the first time, the F-actin-promoted changes in the spatial relationship of strands in the NH2-terminal 25-kDa and COOH-terminal 20-kDa heavy chain fragments of the skeletal myosin subfragment 1 (S-1), detected by their exclusive chemical cross-linking in the rigor F-actin-S-1 complex with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS). Quantitative electrophoretic analysis of the reaction products showed extensive conversion of the 95-kDa heavy chain of the actin-bound S-1 into a new species with an apparent mass of 135 kDa (yield = 50-60%), whereas the heavy chain mobility remained unaffected when actin was omitted. The 135-kDa entity retained the fluorescence of AEDANS-S-1 but not of AEDANS-actin, indicating that it was not a cross-linked acto-heavy chain adduct. Its extent of production depended markedly on the S-1:actin molar ratio and was maximum near a ratio of 1:4. The MBS treatment of acto-S-1 led also to some covalent actin-actin oligomers which could be suppressed by using trypsin-truncated F-actin lacking Cys-374, without altering the generation of the 135-kDa heavy chain derivative. The MBS reaction on the complex of F-actin and tryptic (25-50-20 kDa)-S-1 resulted in a new 40-kDa band, comigrating with actin, which was composed of the N-terminal 25-kDa and C-terminal 20-kDa fragments since it incorporated the fluorescence of the anthroyl group specifically attached to the former peptide and the fluorescence of AEDANS selectively bound to the SH-1 thiol in the latter segment. Blocking SH-1 and SH-2 thiols did not abolish this interfragment cross-linking. However, the addition of millimolar concentrations of MgADP led to its total suppression without dissociation of the acto-S-1 complex as assessed by cosedimentation. Peptide mapping of the 40-kDa adduct indicated that the cross-linking of the 20-kDa region was only to the COOH-terminal stretch of the 25-kDa fragment between amino acids 145 and 204, where resides the flexible gamma-phosphate-binding glycine-rich loop of the S-I ATPase site. The data suggest that the relative movements of the cross-linked heavy chain segments which are reciprocally modulated by F-actin and nucleotides may contribute to the mechanism of interactivity between the actin and ATPase sites of myosin during force generation.