Yeast actin mutants with acidic residues at the N terminus either neutralized (DNEQ) or deleted (delta-DSE) were used to assess the role of N-terminal acidic residues in the interactions of actin with myosin in the contractile cycle. Cosedimentation experiments revealed an approximately 3-fold decrease in the binding constant for DNEQ and delta-DSE actins to myosin subfragment-1 (S1) relative to that of wild type actin both in the presence of MgATP and in the absence of nucleotides (strong binding). DNEQ and delta-DSE actins protected S1 from tryptic digestion as well as the wild type and rabbit actins. The activation of S1 ATPase by DNEQ and delta-DSE actins (up to 50 microM) was very low but increased greatly after cross-linking these mutant actins to S1 by dimethyl suberimidate. Thus, the increased dissociation of mutant actins from S1 in the presence of ATP is the main cause for the low acto-S1 ATPase activities. At low-ionic strength conditions and in the presence of methylcellulose, the DNEQ and delta-DSE actins moved in the in vitro motility assays at a mean velocity similar to that of wild type actin (3.0 microns/s). Yet, the sliding velocity of the N-terminal and D24A/D25A and E99A/E100A mutant actins decreased relative to that of the wild type at all levels of external load introduced into the assay and at low densities of heavy meromyosin (HMM) on the cover slip. This indicates a lower relative force generation with the mutant actins. In contrast, the force generated under the same conditions with the 4Ac mutant actin (with four acidic charges at the N terminus) was higher than with wild type actin. At higher-ionic strength conditions (I = 150 mM), the sliding of the DNEQ and delta-DSE as well as that of the D24A/D25A and E99A/E100A actins ceased even in the presence of methylcellulose, while I341A actin (deficient in strong binding to myosin) still moved. These results indicate the importance of electrostatic actomyosin interactions under physiological salt conditions and show functionally distinct roles for the different myosin binding sites on actin.

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degrees C the ability to activate myosin ATPase at a 100-fold slower rate and unfolded in CD melting experiments at a temperature higher by 11 degrees C than the un-cross-linked actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure but showed a change in the orientation of subdomain 2 and a decrease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major changes upon 50% and 90% ANP cross-linking of F-actin; the Kd and Km values were little affected by the cross-linking, and the Vmax decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decreased the mean speed and the number of sliding filaments in the in vitro motility assays by approximately 35% while the relative force, as measured by using external load in these assays, was inhibited by approximately 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 dimers, trimers, and oligomers. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behavior in the in vitro motility assays. Filaments made of cross-linked dimers moved at approximately 50% of the speed of the un-cross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely, and the oligomer-made 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 process.

Yeast actin mutants with alanines replacing I341 and I345 were studied to assess the role of hydrophobic residues in the alpha-helix 338-348 in interactions with myosin. In structural models of the actomyosin complex, this helix on actin was assigned a prominent role in the strong binding of myosin to actin. Substitution of I341 with alanine reduced the strong binding of actin to myosin subfragment-1 (S1) 9-fold compared to wild-type actin. In addition, the Vmax of the actin-activated S1 ATPase was reduced 4-fold with no change in the Km. In contrast, substitution of I345 with alanine had no significant effect on either the strong binding to S1 or the actin activation of S1 ATPase. The I341A actin filaments were found to slide in the in vitro motility assays at a lower mean velocity (1.6 +/- 0.4 microns/s) than wild-type actin filaments (2.6 +/- 0.3 microns/s). Only 65% of the mutant actin filaments moved in such assays in comparison to 95% of the wild-type filaments. However, addition of 2.0 mM MgADP to the motility assay buffer induced movement of all the I341A filaments at a velocity (1.6 +/- 0.1 microns/s) similar to that of wild-type actin (1.7 +/- 0.1 microns/s). The decrease in motility of the I341A actin filaments in the absence of ADP was attributed to a negative load slowing the mutant filaments and the smaller force produced by the heavy meromyosin and I341A actin system. The latter conclusion was confirmed by showing that a greater percentage of NEM-modified heavy meromyosin (external load) was required for arresting the motion of wild-type actin in the in vitro motility assay than that needed for stopping the I341A filaments.