Single-Molecule Chemo-Mechanical Spectroscopy Provides Structural Identity of Folding Intermediates

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Single-molecule force spectroscopy is crucial for studying biological macromolecule folding.
• Mechanical manipulation offers insights into transient and intermediate states.
• Empirical demarcation has been the primary method for assigning intermediate states.

Purpose of the Study:

• To investigate the role of osmolytes in T4 lysozyme folding and unfolding.
• To explore chemo-mechanical perturbation as a method for characterizing folding intermediates.
• To discriminate between potential structural models of intermediate states.

Main Methods:

• Utilized optical tweezers for single-molecule force spectroscopy on T4 lysozyme.
• Applied chemo-mechanical perturbation by introducing osmolytes (sorbitol, trimethylamine-n-oxide).
• Measured contour length and solvent-accessible surface area during folding trajectories.

Main Results:

• Sorbitol and trimethylamine-n-oxide marginally decelerated unfolding rates.
• These osmolytes specifically modulated early folding events, stabilizing an on-pathway intermediate.
• The study provided two independent metrics (contour length, solvent-accessible surface area) for state analysis.
• Osmolyte-dependent intermediate populations, combined with contour length, allowed discrimination of structural models.

Conclusions:

• Osmolytes can stabilize on-pathway folding intermediates by influencing early folding events.
• Chemo-mechanical perturbation offers a general strategy for structural modeling of equilibrium intermediate states in single-molecule experiments.
• This approach enhances the characterization of transient states in biomolecular folding.