Equilibrium and Non-equilibrium Studies of Protein G using High-Resolution Optical Trapping Unveil Heterogeneous Unfolding Pathways

Abstract

Recent advances in spatial and temporal resolution of biophysical measurements have revealed ‘beyond two-state’ complexity in protein folding, even for small, single-domain proteins. In this work, the folding/unfolding kinetics of the B1 domain of inmmunoglobulin-binding protein G, a well-studied model system, was investigated using high-resolution optical traps. For non-equilibrium force ramp measurements, simple force-dependent folding distributions were observed. However, multiple peaks were seen in the unfolding force distribution, consistent with multiple parallel unfolding pathways or multiple folded states. We then performed long duration fixed trap measurements while slowly stepping the applied force. We were thereby able to directly measure folding and unfolding rate constants versus force under near equilibrium conditions. All molecules showed the same folding behavior, consistent with a simple two-state Bell's formula. However, these same molecules showed one of two unfolding behaviors at low forces, either ‘fast’ and force independent, or ‘slow’ and strongly force dependent. Equilibrium fixed trap observations of individual molecules for over tens of minutes show no switching between unfolding mechanisms amid hundreds of transitions. However, both unfolding mechanisms are observed for the same individual molecules using the force ramp method, indicating that out of equilibrium pulling may be necessary to switch individual molecule unfolding behavior. Taken together, these measurements indicate that protein G unfolds on a more complex energy landscape containing multiple pathways and intermediate states.