Energy landscape of conformational changes for a single unmodified protein

Resolving the free energy landscapes that govern protein biophysics has been obscured by ensemble averaging. While the folding dynamics of single proteins have been observed using fluorescent labels and/or tethers, a simpler and more direct measurement of the conformational changes would not require modifications to the protein. We use nanoaperture optical tweezers to resolve the energy landscape of a single unmodified protein, Bovine Serum Albumin (BSA), and quantify changes in the three-state conformation dynamics with temperature. A Markov model with Kramers’ theory transition rates is used to model the dynamics, showing good agreement with the observed state transitions. This first look at the intrinsic energy landscape of proteins provides a transformative tool for protein biophysics and may be applied broadly, including mapping out the energy landscape of particularly challenging intrinsically disordered proteins.