Probing the structure of F-actin: Cross-links constrain atomic models and modify actin dynamics

Cross-links between protomers in F-actin can be used as a very sensitive pr obe of both the dynamics and structure of F-actin. We have characterized fi laments formed from a previously described yeast actin Q41C mutant, where d isulfide bonds can be formed between the Cys41 that is introduced into subd omain-2 and Cys374 on an adjacent protomer. We find that the distribution o f cross-linked n-mers shows no cooperativity and corresponds to a random pr obability cross-linking reaction. The random distribution suggests that dis ulfide formation does not cause a significant perturbation of the F-actin s tructure. Consistent with this lack of perturbation, three-dimensional reco nstructions of extensively crosslinked filaments, using a new approach to h elical image analysis, show very small structural changes with respect to u ncross-linked filaments. This finding is in conflict with refined models bu t in agreement with the original Holmes et al. model for F-actin. Under con ditions where 94% of the protomers are linked by disulfide bonds, the distr ibution of filament twist becomes more heterogeneous with respect to contro l filaments. A molecular model suggests that strain, introduced by the disu lfide, is relieved by increasing the twist of the long-pitch actin helices. Disulfide formation makes yeast actin filaments similar to three times les s flexible in terms of bending and similar, in this respect, to vertebrate skeletal muscle F-actin. These observations support previous reports that t he rigidity of F-actin can be controlled by the position of subdomain-2, an d that this region is more flexible in yeast F-actin than in skeletal muscl e F-actin. (C) 2001 Academic Press.