Molecular crowding effects on protein stability in a bacterial proteome

TL;DR

This study explores how molecular crowding affects protein stability in bacteria, finding that some proteins become more stable in crowded environments.

Contribution

The study provides new insights into how molecular crowding affects specific subsets of proteins in a bacterial proteome.

Findings

Molecular crowding agents reduced the global mean melting temperature of proteins in Cupriavidus necator.

Some proteins showed increased stability in the presence of crowding agents, particularly hydrophobic or enzyme-like proteins.

The results support a model where crowding agents enhance stability through direct binding or preferential exclusion.

Abstract

Molecular crowding refers to the restriction of space available for molecular mobility in solution due to the presence of other macromolecules. Crowding can influence biological phenomena such as protein stability and folding, diffusion, enzyme kinetics, molecular interactions, and phase separation. However, the relative contributions of entropic (excluded volume) and enthalpic (direct chemical interactions) is still an open question. In contrast to biochemical studies that employ dilute solutions containing a small number of reactants, the cellular environment is considered to be highly crowded, with up to 40% of cell volume occupied by many thousands of proteins and other large biomolecules. We examined the protein stability effects of widely used molecular crowders in a bacterial proteome (Cupriavidus necator) using the Thermal Proteome Profiling (TPP) method, whereby melting temperature is estimated based on protein denaturation and aggregation over a heat gradient. While all six tested compounds (Ficoll 70, Ficoll 400, dextran 40, dextran 86, PEG 1, PEG 8) reduced the global mean melting temperature, significantly increased or decreased stability was observed among distinct subsets of the proteome, with some individual proteins showing sensitivity to multiple crowding agents. Proteins displaying enhanced stability in the presence of crowding agents were more likely to be annotated as hydrophobic, to show classic enzyme-like properties, or propensity for protein interactions. Our data favour a direct binding/preferential exclusion model of enhanced stability, rather than alternative models based on viscosity or crowding.

The online version contains supplementary material available at 10.1038/s41598-026-35990-9.

Figures