Complex folding kinetics of multi-domain proteins disentangled with periodic forces by magnetic tweezers
- 1TU Dresden
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 {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.
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BibTeX
@misc{quintanacatano2026complex,
title = {{Complex folding kinetics of multi-domain proteins disentangled with periodic forces by magnetic tweezers}},
author = {C. A. Quintana-Catano and A. Hartmann and M. Schlierf},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.02.749805},
url = {https://www.biorxiv.org/content/10.64898/2026.10.02.749805v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Complex folding kinetics of multi-domain proteins disentangled with periodic forces by magnetic tweezers
AU - C. A. Quintana-Catano
AU - A. Hartmann
AU - M. Schlierf
PY - 2026
DA - 2026/10/02
PB - bioRxiv
DO - 10.64898/2026.10.02.749805
UR - https://www.biorxiv.org/content/10.64898/2026.10.02.749805v1
AB - 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.
ER -