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bioRxiv · 10.64898/2026.02.16.706251

Chemical interactions in polyethylene glycol-induced condensates lead to an anomalous FRET response from a flexible linker-fluorescent protein crowding sensor

Mohapatra, A.·Antarasen, J.·Latham, D. R.·Barilla, M. A.·Davis, C. M.·Kisley, L.
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Abstract

The cellular cytosol is a crowded environment. Biomolecular Forster resonance energy transfer (FRET) sensors have been developed to measure crowding in cytosol mimics comprised of synthetic polymers such as polyethylene glycol (PEG) and Ficoll that impart an excluded volume effect. In the current study, we explore the unsolicited role of PEG in driving the phase separation of a protein crowding sensor, AcGFP1/mCherry-FRET crowding helix 2 (CrH2), into fluorescent puncta. In contrast, a DNA-based crowding sensor (CrD), with an Alexa488/Cy5 FRET pair, does not form puncta under the same crowding conditions. Using fluorescence recovery after photobleaching imaging, we uncover the liquid-like physical properties of the PEG-induced puncta. Two-color fluorescence microscopy imaging reveals crowder-induced inhomogeneity, concentration variations, and partition coefficient across the dilute and dense phases of the liquid puncta, which remain largely underexplored in bulk fluorometry measurements. Thus, the average crowding sensor response may originate from an aqueous biphasic system, reporting an erroneous average response instead of distinct levels of crowdedness. A comparison of excluded volume effects conferred by Ficoll and PEGs of various molecular weight ranges shows the influence of size, concentration, excluded volume, and chemical composition on the CrH2 sensor response. We demonstrate that PEGs enable phase separation and alter sensor response through a mechanism that may be driven by polymer interactions with the flexible hinge region of CrH2. Overall, we determine the biophysical mechanisms underlying PEG-induced condensation of CrH2 and demonstrate a CrD sensor as an alternative that does not undergo phase separation.

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Mohapatra, A., Antarasen, J., Latham, D. R., Barilla, M. A., Davis, C. M., Kisley, L.. 2026-02-18. Chemical interactions in polyethylene glycol-induced condensates lead to an anomalous FRET response from a flexible linker-fluorescent protein crowding sensor. https://doi.org/10.64898/2026.02.16.706251

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