Full distance-resolved folding energy landscape of one single protein molecule.

Kinetic bulk and single molecule folding experiments characterize barrier properties but the shape of folding landscapes between barrier top and native state is difficult to access. Here, we directly extract the full free energy landscape of a single molecule of the GCN4 leucine zipper using dual beam optical tweezers. To this end, we use deconvolution force spectroscopy to follow an individual molecule's trajectory with high temporal and spatial resolution. We find a heterogeneous energy landscape of the GCN4 leucine zipper domain. The energy profile is divided into two stable C-terminal heptad repeats and two less stable repeats at the N-terminus. Energies and transition barrier positions were confirmed by single molecule kinetic analysis. We anticipate that deconvolution sampling is a powerful tool for the model-free investigation of protein energy landscapes.