Abstract:

Protein function and regulation emerge from the coordinated motion of constituent amino acid residues. While it is routine to determine static structures of proteins and their complexes, direct observation of their functional dynamics is only attainable in special cases. I describe a general approach to observe nonequilibrium protein dynamics, accomplished by applying electrostatic forces to proteins and reading out their structural changes at atomic resolution (electric-field-stimulated time-resolved crystallography, or EF-X). I outline recent methodological developments in data processing using machine learning, which allowed us to conduct multiple variant EF-X experiments on crystals of a human enzyme. These variants comprise 1) replicates, 2) experiments on the same crystal system but at a different orientation of the electric field, and 3) experiments on charge mutants of the same crystal system. Taken together, these measurements allowed us to directly observe how patterns of forces shape protein dynamics and how localized forces propagate through the enzyme backbone. This work sets the stage for direct, general observation of protein allostery.