Complex folding kinetics of multi-domain proteins disentangled with periodic forces by magnetic tweezers

Abstract

Force spectroscopy enables direct investigation of the interplay between free energy and transition kinetics in proteins and nucleic acids. However, multi-domain proteins often display multiple intermediate structures with diverse stabilities and kinetics, making equilibrium conditions difficult to achieve and out-of-equilibrium experiments challenging to interpret. We introduce a single-molecule force spectroscopy assay using periodic forces and high-resolution magnetic tweezers to bridge different stabilities, selectively enhance intermediate populations, and rigorously analyze multi-domain protein folding kinetics at quasi-equilibrium. By applying well-defined periodic square force functions with tunable steady states, we first validate our framework on the two-state folder λ6-85, demonstrating that periodic force experiments can reproduce equilibrium kinetics. Extending this approach to the multi-domain protein mimic (Protein L)8, we show that tuning force, frequency, and amplitude enhances populations of distinct intermediate states. Finally, we dissect the entangled states of the hybrid two-domain protein λ-L, where a 100-fold difference in domain kinetics precludes equilibrium analysis, by expanding from a two-state to a three-state periodic force protocol to direct intermediate populations. Our assay and analysis framework open new possibilities for deciphering complex multi-domain folds and their multi-dimensional folding pathways.