Because steric crowding is most effective when the crowding agent is similar in size to the molecule that it acts upon and the average macromolecule inside cells is much larger than a small protein or peptide, steric crowding is not predicted to affect their folding inside cells. On the other hand, chemical interactions should perturb in-cell structure and stability because they arise from interactions between the surface of the small protein or peptide and its environment. Indeed, previous in vitro measurements of the λ-repressor fragment, λ6-85 , in crowding matrices comprised of Ficoll or protein crowders support these predictions. Here, we directly quantify the in-cell stability of λ6-85 and distinguish the contribution of steric crowding and chemical interactions to its stability. Using a FRET-labeled λ6-85 construct, we find that the fragment is stabilized by 5°C in-cells compared to in vitro. We demonstrate that this stabilization cannot be explained by steric crowding because, as anticipated, Ficoll has no effect on λ6-85 stability. We find that the in-cell stabilization arises from chemical interactions, mimicked in vitro by mammalian protein extraction reagent (M-PER™). Comparison between FRET values in-cell and in Ficoll confirms that U-2 OS cytosolic crowding is reproduced at macromolecule concentrations of 15% w/v. Our measurements validate the cytomimetic of 15% Ficoll and 20% M-PER™ that we previously developed for protein and RNA folding studies. However, because the in-cell stability of λ6-85 is reproduced by 20% v/v M-PER™ alone, we predict that this simplified mixture could be a useful tool to predict the in-cell behaviors of other small proteins and peptides.
Keywords: chemical interactions; fluorescence resonance energy transfer (FRET); lambda repressor (λ6-85); laser-induced temperature jump; macromolecular crowding; protein folding; thermal denaturation.
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