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

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Filamentous actin (F-actin) structure and dynamics are crucial for cellular functions.
• Cross-linking protomers in F-actin offers a sensitive method to probe its structural and dynamic properties.

Purpose of the Study:

• To characterize cross-linked yeast actin filaments using a Q41C mutant.
• To investigate the impact of disulfide bond formation on F-actin structure, dynamics, and flexibility.

Main Methods:

• Utilized a yeast actin Q41C mutant for disulfide bond formation between adjacent protomers.
• Employed a novel helical image analysis approach for 3D reconstructions of cross-linked filaments.
• Analyzed the distribution of cross-linked n-mers and filament twist heterogeneity.

Main Results:

• Disulfide cross-linking distribution was random, indicating no cooperativity and minimal structural perturbation.
• 3D reconstructions showed minor structural changes in cross-linked versus uncross-linked filaments.
• Extensive cross-linking (94%) led to heterogeneous filament twist distribution and increased bending rigidity (approx. 3x).

Conclusions:

• Disulfide formation in yeast F-actin does not significantly perturb overall structure but increases rigidity.
• The findings support the Holmes et al. model and suggest strain relief via increased helix twist.
• Yeast F-actin exhibits greater flexibility than vertebrate skeletal muscle F-actin, controllable by subdomain-2 positioning.