Folding Dynamics of B1 Domain of Protein G using Single Molecule Force Spectroscopy

Abstract

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 final folding step at ∼1ms which depends on how folding is observed. Since protein folding is still a poorly understood process, different denaturants have been put into experiment to better understand the folding process of various proteins. 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. Results presented here show that since GB1 structure undergoes rapid switching between intermediate states of varied extension, thus a two-state model is not appropriate for understanding full folding path of this domain.