{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T10:08:51Z","timestamp":1785146931151,"version":"3.55.0"},"posted":{"date-parts":[[2026,7,23]]},"group-title":"Molecular Biology","reference-count":39,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T00:00:00Z","timestamp":1784764800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,23]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>Weak, transient molecular interactions are ubiquitous amongst biological molecules. Regulatability by intracellular concentrations, localization, physical properties of the cytoplasm, and other factors is central to their importance. Here, we measure co-localization between fluorescently labeled proteins, Miro2 and \u03b2-Pix, to assess their effective binding affinity and its dependence on protein concentration. Co-localization between the two is quantifiable due to the mitochondrial localization of Miro2, which allows the otherwise cytosolic \u03b2-Pix to be drawn to mitochondria. Determining the fraction of overexpressed \u03b2-Pix co-localizing with mitochondria as a function of overexpressed Miro2 levels allows calculation of an effective dissociation constant from a mass-action binding model. Both the fit to the model and the effective dissociation constant for Miro2 and \u03b2-Pix binding depend on the concentration of \u03b2-Pix, which suggests that one or more model assumptions do not hold. Additional experiments reveal that competitive binding and\/or \u03b2-Pix sequestration by Git1 are possible explanations for the \u03b2-Pix concentration dependence. Our modeling strategy is readily extendable to ectopic localization of proteins to mitochondria and other compartments for the assessment of co-localization with a binding partner. This reveals fundamental properties of protein-protein interactions and how they may be regulated by protein concentration and other intracellular conditions.<\/jats:p>","DOI":"10.64898\/2026.07.20.739652","type":"posted-content","created":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T12:00:14Z","timestamp":1784894414000},"source":"Crossref","is-referenced-by-count":0,"title":["A co-overexpression strategy allows effective protein-protein binding affinities to be assessed as a function of concentration within live cells"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8360-762X","authenticated-orcid":false,"given":"Samantha","family":"Stam","sequence":"first","affiliation":[{"name":"Department of Molecular and Cellular Biology, University of California Davis, Davis, California"},{"name":"James Franck Institute, University of Chicago, Chicago, 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