"The B1 domain of protein G is a well-studied model system of protein folding that has a mixed secondary structure, high stability and relatively slow folding kinetics. However, recent measurements on the sub-millisecond timescale show significant complexity before the final folding step at 1m0303s, which depends on how folding is observed. Applying force is one natural method for denaturing proteins in order to observe the process of refolding in detail, but the time resolution of standard instruments typically make investigation of fast folding proteins difficult. In this study, two alkyne-containing unnatural amino acid residues were added to both N and C termini of GB1 and connected via click chemistry to double stranded DNA handles. The protein-DNA chimeras were then suspended between a pair of polystyrene beads held in high-resolution dual optical traps and reversible folding/unfolding events were observed both during force-extension pulling/relaxation experiments as well as under constant force feedback conditions. Presented results for standard, out-of-equilibrium force ramp measurements of GB1 show transitions between two apparently unique folded and unfolded states in agreement with polymer modeling and simple two-state modeling. However, force distributions suggest the presence of intermediate states or multiple folding pathways. Long duration fixed trap position measurements directly reveal equilibrium folding and unfolding reactions and confirm complexity in unfolding and the presence of intermediates. For GB1, while the folding rates are consistent over many molecules and depend strongly on force, the unfolding rates vary widely between molecules."--Page ii.
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