Method to extract multiple states in F₁-ATPase rotation experiments from jump distributions

Area of Science:

• Biophysics
• Enzyme kinetics
• Single-molecule biophysics

Background:

• F1-adenosinetriphosphatase (F1-ATPase) is a molecular motor crucial for cellular energy production.
• Understanding the rapid dynamics of F1-ATPase is essential for elucidating its catalytic mechanism.
• Existing methods like the "stalling" technique provide valuable but incomplete insights into F1-ATPase function.

Purpose of the Study:

• To develop and validate a novel method for analyzing fast single-molecule rotation trajectories in F1-ATPase.
• To identify transient states and conformational changes not readily detectable by other techniques.
• To complement existing methods and provide a more comprehensive understanding of F1-ATPase's catalytic cycle.

Main Methods:

• Analysis of fast (10 μs) single-molecule rotation trajectories.
• Utilizing the distribution of angular jumps during transitions between catalytic dwells.
• Application of multistate theory incorporating viscoelastic fluctuations of the imaging probe.

Main Results:

• A bimodal distribution of angular jumps was observed, indicating the presence of multiple states at the same rotation angle.
• A theoretical profile of angular jumps was predicted using a 3-state model, showing good agreement with experimental data.
• Inclusion of a fourth transient state (∼10 μs lifetime) improved agreement, suggesting a rapid ATP binding and ADP release transient.

Conclusions:

• The proposed method effectively reveals transient states in F1-ATPase, complementing existing techniques.
• A transient state with a lifetime of approximately 10 μs was identified, significantly shorter than previously characterized ADP release events.
• The findings provide a more detailed mechanistic understanding of the F1-ATPase catalytic cycle, particularly the interplay of ATP binding and ADP release.