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

This study reveals that polyethylene glycol (PEG) induces liquid-liquid phase separation in a protein-based FRET crowding sensor through specific chemical interactions with its flexible linker, leading to anomalous FRET signals and erroneous bulk measurements, whereas a DNA-based sensor remains unaffected by this condensation mechanism.

The Gist

The Big Picture: A Crowded Room and a Broken Thermometer

Imagine a cell is like a very crowded party room. It's packed with people (proteins, DNA, and other molecules) so tightly that there is almost no empty space left. Scientists call this "macromolecular crowding."

To understand how crowded this room is, scientists invented a special "thermometer" called a FRET sensor.

• How it works: Imagine a person holding two balloons (one green, one red) connected by a stretchy rubber band.
  • If the room is empty, the person can stretch the rubber band out, keeping the balloons far apart.
  • If the room gets crowded, people bump into the person, forcing them to curl up. The rubber band shortens, and the balloons get closer together.
  • When the balloons get close, the green one "talks" to the red one, changing the color of the light they give off. By measuring this color change, scientists can tell how crowded the room is.

The Problem: The "Crowding" Agent Caused a Meltdown

In this study, the scientists used a common substance called PEG (Polyethylene Glycol) to simulate a crowded room. They thought PEG was just an inert filler, like adding more people to the party to make it crowded.

They used two different types of "thermometers":

1. CrH2 (The Protein Sensor): Made of proteins and a stretchy, flexible rubber band.
2. CrD (The DNA Sensor): Made of DNA and a rigid structure.

What happened?
When they added PEG to the CrH2 sensor, something weird happened. Instead of just getting crowded and curling up, the sensors suddenly clumped together into tiny, glowing droplets (called condensates or puncta).

It's as if, instead of just getting squeezed by the crowd, the people in the room suddenly decided to hold hands and form a giant, sticky ball in the middle of the floor.

• The DNA sensor (CrD) behaved normally. It just got squeezed and reported the crowd level accurately.
• The Protein sensor (CrH2) got stuck in these glowing balls.

The Investigation: Why Did the Protein Sensor Clump?

The scientists wanted to know: Is the sensor just measuring the crowd, or is the crowd (PEG) actually changing the sensor's behavior?

They discovered three major things:

1. It's a Liquid, Not a Solid
Using a technique called FRAP (which is like shining a bright light to "bleach" a spot and watching if the color comes back), they found that these glowing balls are liquid.

• Analogy: Imagine a drop of honey. If you poke it, it flows. If you poke a rock, it stays still. The sensor droplets flowed and reformed, meaning the sensors were trapped in a liquid-like state, not a solid clump.

2. The "Average" Reading is a Lie
When scientists measure these sensors in a test tube, they usually look at the whole mixture and get an "average" number.

• The Trap: Because the sensors are hiding inside the sticky droplets (where it's super crowded) and the empty space around them (where it's less crowded), the "average" reading is wrong. It's like trying to measure the temperature of a room by averaging the heat of a campfire and an ice cube. You get a "warm" number, but that doesn't tell you the truth about either the fire or the ice.

3. It's Not Just About Size (The "Excluded Volume" Myth)
Scientists used to think crowding was just about size (like trying to fit a big suitcase into a small car). They thought PEG worked just by taking up space.

• The Discovery: They compared PEG to another crowder called Ficoll (which is bigger but chemically different). Ficoll crowded the room but didn't make the sensors clump. PEG made them clump.
• The Real Reason: PEG isn't just a space-filler; it's chemically "sticky" to the protein's flexible rubber band. It interacts with the sensor's "hinge" (the stretchy part), causing it to lose its shape and stick to other sensors.

The Conclusion: A Warning for Scientists

The paper concludes with a very important warning for anyone studying cells:

• Don't trust the "average": If you use protein-based sensors with PEG, you might be measuring a phase separation (clumping) rather than just crowding. Your data could be misleading.
• Choose your tools wisely: If you want to measure crowding without the sensors clumping up, the DNA-based sensor (CrD) is a much better choice because it stays dissolved and doesn't react chemically with PEG.
• Look closer: You can't just look at the whole test tube; you need to use a microscope to see if your sensors are forming hidden droplets.

In short: The "crowding" agent (PEG) didn't just squeeze the sensor; it chemically tricked the protein sensor into forming liquid droplets, giving a false reading of how crowded the environment really is. The DNA sensor, however, stayed calm and told the truth.

Technical Summary

1. Problem Statement

Macromolecular crowding is a critical factor in cellular physiology, influencing protein folding, assembly, and dynamics. To study this, researchers use Förster Resonance Energy Transfer (FRET)-based sensors that change conformation in response to excluded volume effects.

• The Issue: Synthetic crowders like Polyethylene Glycol (PEG) are widely used to mimic intracellular crowding. However, PEG is known to induce phase separation in proteins, a phenomenon distinct from simple excluded volume effects.
• The Gap: It was previously unclear whether the FRET response observed in protein-based crowding sensors (specifically the CrH2 sensor) in the presence of PEG was a true measure of crowding-induced compaction or an artifact caused by the sensor undergoing liquid-liquid phase separation (LLPS) into condensates. Furthermore, bulk fluorometry measurements often mask spatial heterogeneity, potentially reporting erroneous average values.

2. Methodology

The study employed a comparative approach using two distinct crowding sensors and multiple biophysical characterization techniques:

• Sensors Used:
  • CrH2: A protein-based sensor consisting of AcGFP1 and mCherry linked by a flexible hinge containing rigid $\alpha$-helical motifs and disordered regions.
  • CrD: A DNA-based sensor (Alexa488/Cy5 FRET pair) with a rigid double-stranded backbone, serving as a control to test if phase separation is specific to the protein architecture.
• Crowding Agents:
  • PEG: Various molecular weights (1, 8, 20, 35 kDa) were used to induce crowding.
  • Ficoll PM 70: Used as a control crowder known to exert excluded volume effects without inducing phase separation in this context.
• Experimental Techniques:
  • Two-Color Fluorescence Microscopy: To visualize spatial distribution, puncta formation, and calculate partition coefficients ($K_p$) between dilute and dense phases.
  • Fluorescence Recovery After Photobleaching (FRAP): To determine the liquid-like properties and diffusion dynamics of the formed condensates.
  • Fluorescence Spectroscopy: To measure bulk FRET ratios (Donor/Acceptor) and monitor intensity changes.
  • Circular Dichroism (CD) Spectroscopy: To assess secondary structural changes (specifically $\alpha$-helical content) in the presence of PEG.
  • Ultracentrifugation: To ensure samples were free of pre-formed aggregates before imaging.

3. Key Contributions

• Discovery of Anomalous Phase Separation: The authors demonstrated that the CrH2 protein sensor undergoes liquid-liquid phase separation (forming fluorescent puncta) in the presence of PEG, whereas the DNA-based CrD sensor and CrH2 in the presence of Ficoll do not.
• Differentiation of Crowding Mechanisms: The study proved that the FRET response in PEG is not solely driven by excluded volume effects but is significantly influenced by specific chemical interactions between PEG and the sensor's flexible linker.
• Methodological Warning: The paper highlights that bulk fluorometry can yield misleading "average" FRET ratios when phase separation occurs, as it conflates signals from the dilute phase and the dense condensate phase.
• Alternative Sensor Validation: The study validates the DNA-based CrD sensor as a robust alternative for in vitro crowding studies involving PEG, as it resists phase separation.

4. Results

• Phase Separation and Puncta Formation:

  • CrH2 formed distinct, round, fluorescent puncta in the presence of PEG (concentration-dependent, starting around 180 mg/mL for 8 kDa PEG).
  • No puncta were observed with Ficoll or with the CrD sensor under identical conditions.
  • FRAP analysis confirmed these puncta are liquid condensates with rapid fluorescence recovery, indicating dynamic exchange of molecules.

• Anomalous FRET Response:

  • Bulk vs. Microscopy: While bulk fluorometry showed a non-linear decrease in the Donor/Acceptor (D/A) ratio (suggesting compaction), microscopy revealed this was due to the sensor partitioning into dense phases.
  • Intensity Anomalies: In the presence of PEG, the donor (AcGFP1) intensity initially increased at low PEG concentrations (100 mg/mL) before decreasing, a phenomenon not seen with Ficoll. This suggests PEG alters the photophysics or local refractive index of the fluorophore.
  • Partitioning: The partition coefficient ($K_p$) increased with PEG concentration, showing significant enrichment of CrH2 in the dense phase compared to the dilute phase.

• Role of Excluded Volume vs. Chemical Interactions:

  • Calculations of excluded volume fractions showed that PEG 1 kDa (smaller excluded volume) caused a massive change in the FRET ratio, comparable to larger PEGs, while Ficoll (large excluded volume) caused a linear, moderate response.
  • This indicates that excluded volume is not the singular driving force; specific chemical interactions are required for the observed phase separation.

• Structural Changes (CD Spectroscopy):

  • CD spectra revealed a loss of $\alpha$-helical content in CrH2 upon the addition of PEG (specifically between 100–200 mg/mL), coinciding with the phase transition point observed in microscopy.
  • The authors hypothesize that PEG interacts with the flexible, helical linker region of CrH2, destabilizing the helix and promoting intermolecular interactions that drive condensation.

5. Significance

• Re-evaluation of Crowding Studies: The findings necessitate a re-evaluation of previous studies using PEG and protein-based FRET sensors. Data reporting "crowding" in PEG may actually be reporting phase separation artifacts.
• Sensor Design Guidelines: The study emphasizes the need for careful sensor design. Rigid backbones (like DNA in CrD) may be preferable for studying pure excluded volume effects in PEG, while protein-based sensors with flexible linkers are prone to phase separation.
• Biological Implications: Since intracellular environments are complex, researchers must use microscopy to verify that sensors are not forming condensates in live cells, as this would confound measurements of macromolecular crowding.
• Mechanistic Insight: The work provides a biophysical mechanism where chemical interactions (enthalpic) between PEG and specific protein domains (helical linkers) can override simple steric exclusion (entropic) to drive phase separation.

In conclusion, the paper establishes that PEG-induced condensation of the CrH2 sensor creates an anomalous FRET response driven by chemical interactions and phase separation rather than simple crowding, urging the scientific community to adopt microscopy-based validation and alternative sensor designs for accurate in vitro crowding measurements.