Complex pathways in folding of protein G explored by simulation and experiment

Area of Science:

• Biophysics
• Protein dynamics
• Computational biology

Background:

• The B1 domain of protein G is a well-studied model for protein folding.
• Previous experimental evidence for folding intermediates was limited to slow kinetics and few probes.

Purpose of the Study:

• To investigate protein G folding on the submillisecond timescale.
• To compare experimental observations with computational models.

Main Methods:

• Utilized microfluidic mixers for submillisecond kinetic measurements.
• Employed multiple probes: tryptophan fluorescence, circular dichroism, and photochemical oxidation.
• Constructed a Markov State Model from large-scale molecular dynamics simulations.

Main Results:

• Observed protein folding kinetics on the submillisecond timescale.
• Different probes exhibited distinct folding kinetics.
• The Markov State Model revealed a complex network of states.

Conclusions:

• Protein G folding involves numerous pathways preceding the final step.
• These folding pathways do not appear to have significant free energy barriers.
• The complexity of folding pathways is dependent on the observable probes used.