Intrastrand cross-linked actin between Gln-41 and Cys-374. III. Inhibitionof motion and force generation with myosin

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitr ophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-3 74 on adjacent monomers, were examined for several preparations of such act in. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lo st at 60 degrees C the ability to activate myosin ATPase at a 100-fold slow er rate and unfolded in CD melting experiments at a temperature higher by 1 1 degrees C than the un-cross-linked actin. Electron microscopy and image r econstruction of these filaments did not reveal any gross changes in F-acti n structure but showed a change in the orientation of subdomain 2 and a dec rease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major chang es upon 50% and 90% ANP cross-linking of F-actin; the K-d and K-m values we re little affected by the crosslinking, and the V-max decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decrea sed the mean speed and the number of sliding filaments in the in vitro moti lity assays by similar to 35% while the relative force, as measured by usin g external load in these assays, was inhibited by similar to 25%. The mean speed of actin filaments decreased with the increase in their cross-linking and approached 0 for the 90% cross-linked actin. Also examined were actin filaments reassembled from cross-linked and purified ANP cross-linked dimer s, trimers, and oligomers. All of these filaments had the same acto-S1 ATPa se and rigor SI binding properties but different behavior in the in vitro m otility assays. Filaments made of cross-linked dimers moved at similar to 5 0% of the speed of the un-cross-linked actin. The movement of filaments mad e of cross-linked trimers was inhibited more severely, and the oligomer-mad e filaments did not move at all. These results show the uncoupling between force generation and other events in actomyosin interactions and emphasize the role of actin filament structure and dynamics in the contractile proces s.