Modular Engineering of Thermo-Responsive Allosteric Proteins

Hoffmann, K. H.; Kroell, A.-S.; Motzkus, N. A.; Lemmen, N.; Happ, N.; Wolf, B.; von Bachmann, A.; Southern, N.; Vogd, F.; Aschenbrenner, S.; Niopek, D.; Mathony, J.

doi:10.1101/2025.05.02.651844

bioRxiv · 10.1101/2025.05.02.651844

Modular Engineering of Thermo-Responsive Allosteric Proteins

Hoffmann, K. H.·Kroell, A.-S.·Motzkus, N. A.·Lemmen, N.·Happ, N.·Wolf, B.·von Bachmann, A.·Southern, N.·Vogd, F.·Aschenbrenner, S.·Niopek, D.·Mathony, J.

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Abstract

Thermogenetics enables non-invasive spatiotemporal control over protein activity in living cells and tissues, yet its applications have largely been restricted to transcriptional regulation and membrane recruitment. Here, we present a generalizable strategy for engineering thermosensitive allosteric proteins through the insertion of optimized Avena sativa LOV2 domain variants. Applying this approach to a diverse set of structurally and functionally unrelated proteins in Escherichia coli, we generated potent, thermo-switchable chimeric variants that can be tightly controlled within narrow temperature ranges (37-41{degrees}C). Extending this strategy to mammalian systems, we engineered the first CRISPR-Cas genome editors directly modulated by subtle temperature changes within the physiological range. Finally, we showcase the incorporation of a chemoreceptor domain as an alternative thermosensing module, suggesting thermo-sensitivity to be a widespread feature in receptor domains. This work expands the toolkit of thermogenetics, providing a blueprint for temperature-dependent control of virtually any protein of interest.

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Hoffmann, K. H., Kroell, A.-S., Motzkus, N. A., Lemmen, N., Happ, N., Wolf, B., von Bachmann, A., Southern, N., Vogd, F., Aschenbrenner, S., Niopek, D., Mathony, J.. 2025-05-02. Modular Engineering of Thermo-Responsive Allosteric Proteins. https://doi.org/10.1101/2025.05.02.651844

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