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 probe of both the dynamics and structure of F-actin. We have characterized filaments formed from a previously described yeast actin Q41C mutant, where disulfide bonds can be formed between the Cys41 that is introduced into subdomain-2 and Cys374 on an adjacent protomer. We find that the distribution of cross-linked n-mers shows no cooperativity and corresponds to a random probability cross-linking reaction. The random distribution suggests that disulfide formation does not cause a significant perturbation of the F-actin structure. Consistent with this lack of perturbation, three-dimensional reconstructions of extensively cross-linked filaments, using a new approach to helical image analysis, show very small structural changes with respect to uncross-linked filaments. This finding is in conflict with refined models but in agreement with the original Holmes et al. model for F-actin. Under conditions where 94 % of the protomers are linked by disulfide bonds, the distribution of filament twist becomes more heterogeneous with respect to control filaments. A molecular model suggests that strain, introduced by the disulfide, is relieved by increasing the twist of the long-pitch actin helices. Disulfide formation makes yeast actin filaments approximately three times less flexible in terms of bending and similar, in this respect, to vertebrate skeletal muscle F-actin. These observations support previous reports that the rigidity of F-actin can be controlled by the position of subdomain-2, and that this region is more flexible in yeast F-actin than in skeletal muscle F-actin.
Orlova
A
A
Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, VA 22908-0733, USA.
Galkin
V E
VE
VanLoock
M S
MS
Kim
E
E
Shvetsov
A
A
Reisler
E
E
Egelman
E H
EH
D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D003850:Deoxyribonuclease I / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D008958:Models, Molecular; D015233:Models, Statistical; D018482:Muscle, Skeletal / Q000737:chemistry; D009154:Mutation; D011487:Protein Conformation; D015003:Yeasts / Q000737:chemistry
D000199:Actins; D004220:Disulfides; D003850:Deoxyribonuclease I