Keywords

IDP, Crowded Environments, Single-Molecule FRET, Polymer Physics, Conformational Dynamics

Reference

DOI: https://doi.org/10.1073/pnas.1322611111

Abstract

Intrinsically disordered proteins (IDPs), due to their high flexibility and lack of stable tertiary structure, are especially sensitive to molecular crowding in cellular environments.
Using single-molecule Förster resonance energy transfer (FRET), this study quantifies how crowding agents (modeled by biocompatible polymers like PEG) affect IDP conformations.
Compaction of IDPs occurs with both increasing concentration and size of crowders, contradicting scaled-particle theory predictions. Instead, data are consistent with polymer-specific models where both IDPs and crowding agents are treated as penetrable polymers.
Results highlight excluded volume interactions, polymeric effects, and suggest a need to revise standard crowding paradigms for IDPs.

Notes

1. General Summary

  • IDPs often alter their dimensions under crowded conditions, though some remain disordered without gaining structure.
  • PEG polymers used as crowding agents because of their uncharged nature, high solubility, and availability in a wide range of sizes (from monomers to high polymers).
  • Focus on how IDP conformation (especially radius of gyration, Rg) responds to crowder concentration and size.

2. Methodological Approach

  • Single-molecule FRET between Alexa Fluor 488 (donor) and Alexa Fluor 594 (acceptor) labels attached via engineered cysteines to monitor end-to-end distance of IDPs.
  • Used Flory-Fisk distribution to analyze chain conformations under different conditions.
  • Investigated four representative IDPs:
    • Prothymosin-α N-terminal and C-terminal segments (ProTα-N, ProTα-C) — highly charged, remain unfolded.
    • ACTR — binding domain of thyroid hormone/retinoid receptor activator, IDP that folds upon partner binding.
    • IN (HIV-1 integrase N-terminal domain) — folds upon ligand binding.

3. Core Findings and Insights

  • Increasing crowder concentration leads to IDP compaction, observable as reduced Rg via FRET.
  • Larger crowders cause more compaction than smaller ones, at odds with scaled-particle theory, which predicts larger crowders should create larger interstitial spaces.
  • IDPs collapse more with increasing size of the crowders — a surprising and key observation challenging classical models.
  • Introduction of “Gaussian cloud” model:
    • IDPs treated as Gaussian-distributed monomer clouds.
    • Small crowders penetrate the cloud easily with minimal perturbation.
    • Larger crowders cannot penetrate without steric clashes, leading to IDP compaction.
  • Two regimes identified:
    1. Short chain regime: IDP compaction due to loss of free volume as crowders enter the chain’s exploration space.
    2. Long chain regime: At high concentrations, crowders overlap, influencing IDPs via polymer-polymer interpenetration effects.

4. Experiments and Controls

  • Different PEG sizes used to systematically study crowder size effects.
  • Crowder-induced compaction is observed regardless of IDP type, though extent varies.
  • Salt and denaturant additions do not prevent compaction, indicating dominance of hard-core repulsion over weak nonspecific interactions.
  • Tests with other polymers confirm general behavior, suggesting this is a robust polymeric effect, not specific to PEG.
  • Hard-core repulsion and polymer physics, rather than specific chemical interactions, dominate IDP responses to crowding.

5. Broader Implications

  • Standard models (like scaled-particle theory) are insufficient to explain IDP responses to crowding.
  • The polymer nature of both IDPs and crowders must be accounted for in understanding cellular crowding effects.
  • Excluded volume interactions and interpenetrability between flexible polymers emerge as key factors in cellular-like environments.
  • Crowding may dynamically influence IDP-mediated signaling by modulating IDP size and accessibility.
  • Important for understanding cellular regulation of IDP functions, including partner binding and phase separation.

6. RD’s Thoughts and Learnings

  • Super interesting that crowder size, not just concentration, controls IDP compaction.
  • Reinforces the idea that IDPs are not static coils but responsive polymers influenced by their surroundings.
  • “Gaussian cloud” model offers a conceptually intuitive way to think about IDP-crowder interactions — love this model.
  • Seeing IDPs as “soft polymers” rather than rigid bodies makes way more sense — fits into emerging views on IDP biophysics.
  • Cool to see experimental work (FRET) combined with physical models to challenge prevailing ideas like scaled-particle theory.
  • Potential to explore how PTMs might alter these crowding responses — e.g., phosphorylation could change how IDPs behave in dense environments.

Take-home Messages

  • IDP conformations are highly sensitive to molecular crowding, responding to both crowder size and concentration.
  • Compaction increases with crowder size, a finding that challenges classic hard-sphere-based models.
  • The “Gaussian cloud” model explains why larger crowders compact IDPs more strongly — penetration probability drops with size.
  • Polymer-specific effects, including interpenetrability and excluded volume, are essential to understanding IDP behavior in cells.
  • These insights reshape our understanding of IDP dynamics in crowded environments — essential for grasping how IDPs work in real cells.

RD finds the Gaussian cloud model super inspiring — finally, a polymer physics view that makes sense for IDPs!✨