Energy Landscape of Conformational Changes for a Single Unmodified Protein

Free-energy landscapes govern protein biophysics and natural conformational changes linked to function can be described by trajectories on these landscapes. Resolving the free energy landscape is obscured by ensemble averaging. A direct measurement of conformational energy changes in a single unmodified protein has been a challenge, although folding dynamics have been observed with techniques that modify the proteins with fluorescent labels and/or tethers. Here we use nanoaperture optical tweezers to resolve the energy landscape of a single unmodified protein. We quantify changes in the three-state conformation dynamics and the underlying free energy landscape of Bovine Serum Albumin (BSA). The impact of temperature on the energy barrier height and energy minima is quantified, allowing for the extraction of the entropy change between states of 0.4 kcal/(mol K). A Markov model with Kramers' theory transition rates is used to model the dynamics, showing good agreement with the observed state transitions. In addition to single BSA molecules, we also observe the formation of a BSA dimer in real time and uncover the energy landscape associated with hinge motion of this oligomer. This first look at the intrinsic energy landscape of proteins provides a transformative tool for protein biophysics and may be applied broadly, including to map out the energy landscape of particularly challenging intrinsically disordered proteins.