Out-of-Equilibrium Biophysical Chemistry: The Case for Multidimensional, Integrated Single-Molecule Approaches

[Image: see text] Out-of-equilibrium processes are ubiquitous across living organisms and all structural hierarchies of life. At the molecular scale, out-of-equilibrium processes (for example, enzyme catalysis, gene regulation, and motor protein functions) cause biological macromolecules to sample an ensemble of conformations over a wide range of time scales. Quantifying and conceptualizing the structure–dynamics to function relationship is challenging because continuously evolving multidimensional energy landscapes are necessary to describe nonequilibrium biological processes in biological macromolecules. In this perspective, we explore the challenges associated with state-of-the-art experimental techniques to understanding biological macromolecular function. We argue that it is time to revisit how we probe and model functional out-of-equilibrium biomolecular dynamics. We suggest that developing integrated single-molecule multiparametric force–fluorescence instruments and using advanced molecular dynamics simulations to study out-of-equilibrium biomolecules will provide a path towards understanding the principles of and mechanisms behind the structure–dynamics to function paradigm in biological macromolecules.