{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "TU Dresden"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 3,
  "authors": [
    {
      "name": "C. A. Quintana-Catano"
    },
    {
      "name": "A. Hartmann"
    },
    {
      "affs": [
        0
      ],
      "name": "M. Schlierf"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/0w9hkdw55s02d.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.02.749805",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.02.749805v1.full",
  "id": "0w9hkdw55s02d",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.02.749805v1",
  "language": null,
  "licence": "CC-BY-NC",
  "licence_label": "CC BY-NC",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "biophysics"
    ],
    [
      "openalex",
      "1312|Protein Structure and Dynamics"
    ]
  ],
  "openalex": "W7219586945",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.02.749805v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-02",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/0w9hkdw55s02d.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:25:57Z",
      "native_id": "10.64898/2026.10.02.749805",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.02.749805v2"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "biorxiv:biophysics",
      "subfield": "biophysics"
    }
  ],
  "title": "Complex folding kinetics of multi-domain proteins disentangled with periodic forces by magnetic tweezers",
  "title_text": "Complex folding kinetics of multi-domain proteins disentangled with periodic forces by magnetic tweezers",
  "type": "preprint",
  "updated_at": "2026-10-30T12:26:09Z",
  "url": "https://research.draconitic.com/paper/0w9hkdw55s02d",
  "venue": "bioRxiv",
  "versions": []
}