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Polyethylene Glycol Induced Condensation Leads to an Anomalous FRET Response from a Flexible Linker-Fluorescent Protein Crowding Sensor
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DOI：10.1021/acssensors.5c03872.png)
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Abstract
En 中文
The cellular cytosol is a crowded environment. Biomolecular Förster resonance energy transfer (FRET) sensors have been developed to measure crowding in cytosol mimics composed of synthetic polymers such as polyethylene glycol (PEG) and Ficoll that impart an excluded volume effect. We investigate the PEG-driven phase separation of a protein crowding sensor, AcGFP1/mCherry-FRET crowding helix 2 (CrH2), into fluorescent puncta compared to a DNA-based crowding sensor (CrD) with an Alexa488/Cy5 FRET pair that did not form observable puncta under the same crowding conditions of 100−400 mg/mL 8 kDa PEG. 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 coefficients 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 weights shows the influence of size, concentration, excluded volume, and chemical composition on the CrH2 sensor response. We demonstrate that ≥18% PEG (w/v) was sufficient to enable the phase separation of CrH2 and alter sensor response through a mechanism that may be driven by polymer interactions with the flexible hinge region. We also show that CrD can form fluorescence puncta upon charge-neutralization with poly-l-lysine. Thus, our study emphasizes the need to probe crowding environments with orthogonal sensors.
Keywords:
Fluorescence
Fluorescence resonance energy transfer
Peptides and proteins
Phase separation
Sensors
molecular sensor
protein crowding sensor
DNA-based sensor
macromolecular crowding
FRET
intermolecular FRET
condensate
phase separation
helix
flexible linker
AcGFP1/mCherry-FRET
Journal
IF:
9.1
Papers:
975
Citations:
2.6W
Organization
Cited Papers