Mapping fast protein folding with multiple-site fluorescent probes.

Area of Science:

• Protein dynamics
• Biophysics
• Computational biology

Background:

• Fast protein folding is complex, with limited structural detail from current experimental methods.
• Microsecond folding experiments offer low-resolution insights into protein dynamics.
• The five-helix bundle protein λ6-85 serves as a model system for studying folding mechanisms.

Purpose of the Study:

• To enhance the structural resolution of protein folding experiments.
• To investigate the folding dynamics of the λ6-85 protein using multiple probes.
• To compare experimental results with all-atom molecular dynamics simulations for mechanistic validation.

Main Methods:

• Engineering of three fluorescent tryptophan-tyrosine contact probes into the λ6-85 protein.
• Temperature jump relaxation experiments to measure kinetic timescales of helix contacts.
• All-atom molecular dynamics simulations to analyze protein folding trajectories.
• Autocorrelation analysis and time-smoothing of simulation data.

Main Results:

• Experimental data revealed two distinct kinetic timescales for helix contact formation (1-3 vs. 1-2/3-2).
• Molecular dynamics simulations reproduced these distinct timescales, confirming a robust folding mechanism.
• Simulations suggested helix 2 misfolds, forming a transient off-pathway trap, explaining experimental observations.
• The ordering of kinetic timescales was robust across different simulation analysis methods.

Conclusions:

• Combining fluorescent probes and molecular dynamics simulations provides a powerful tool for studying protein folding.
• The study validates a single folding mechanism with distinct contact formation timescales, rather than multiple mechanisms.
• The findings highlight the utility of multi-parameter comparisons between experiment and simulation for robust mechanistic testing.