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Chemical interactions modulate λ6-85 stability in cells.

Edward Knab1, Caitlin M Davis1

  • 1Department of Chemistry, Yale University, New Haven, Connecticut, USA.

Protein Science : a Publication of the Protein Society
|June 14, 2023
PubMed
Summary
This summary is machine-generated.

Cellular environments stabilize small proteins through chemical interactions, not steric crowding. This study quantifies in-cell protein stability, finding chemical interactions are key, and suggests a simplified in vitro method for predicting protein behavior.

Keywords:
chemical interactionsfluorescence resonance energy transfer (FRET)lambda repressor (λ6-85)laser-induced temperature jumpmacromolecular crowdingprotein foldingthermal denaturation

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Biophysics

Background:

  • Steric crowding's effect on protein folding is size-dependent.
  • Cellular environments present unique challenges for protein stability due to macromolecule size and chemical interactions.
  • Previous in vitro studies suggested chemical interactions, not steric crowding, influence in-cell protein stability.

Purpose of the Study:

  • To directly quantify the in-cell stability of the lambda-repressor fragment (λ6-85).
  • To differentiate the contributions of steric crowding and chemical interactions to protein stability within a cellular context.
  • To validate and refine in vitro models for predicting in-cell protein behavior.

Main Methods:

  • Utilized a Förster Resonance Energy Transfer (FRET)-labeled λ6-85 construct for stability measurements.
  • Compared in-cell stability with in vitro stability in the presence of Ficoll (steric crowder) and mammalian protein extraction reagent (M-PER™, chemical interactions).
  • Assessed macromolecule concentration effects on cellular crowding using FRET values.

Main Results:

  • The λ6-85 fragment exhibited a 5°C stabilization in-cell compared to in vitro conditions.
  • Ficoll, a steric crowder, did not affect λ6-85 stability, confirming steric crowding's minimal role.
  • In-cell stabilization was attributed to chemical interactions, effectively mimicked by M-PER™ in vitro.
  • Cytosolic crowding in U-2 OS cells was cytomimetically reproduced by 15% (w/v) macromolecule concentrations.
  • A simplified in vitro mixture of 20% (v/v) M-PER™ alone accurately reproduced the in-cell stability of λ6-85.

Conclusions:

  • Chemical interactions, rather than steric crowding, are the primary drivers of small protein stabilization within cellular environments.
  • The study validates existing in vitro cytomimetic mixtures for protein and RNA folding studies.
  • A simplified M-PER™-based in vitro system shows promise for predicting the in-cell behavior of other small proteins and peptides.