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

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 {lambda}6-85, demonstrating that periodic force experiments can reproduce equilibrium kinetics. Extending this approach to the multi-domain protein mimic (Protein L), 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 {lambda}-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.