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An in vitro mimic of in-cell solvation for protein folding studies.

ABSTRACT: Ficoll, an inert macromolecule, is a common in vitro crowder, but by itself it does not reproduce in-cell stability or kinetic trends for protein folding. Lysis buffer, which contains ions, glycerol as a simple kosmotrope, and mimics small crowders with hydrophilic/hydrophobic patches, can reproduce sticking trends observed in cells but not the crowding. We previously suggested that the proper combination of Ficoll and lysis buffer could reproduce the opposite in-cell folding stability trend of two proteins: variable major protein-like sequence expressed (VlsE) is destabilized in eukaryotic cells and phosphoglycerate kinase (PGK) is stabilized. Here, to discover a well-characterized solvation environment that mimics in-cell stabilities for these two very differently behaved proteins, we conduct a two-dimensional scan of Ficoll (0-250 mg/ml) and lysis buffer (0-75%) mixtures. Contrary to our previous expectation, we show that mixtures of Ficoll and lysis buffer have a significant nonadditive effect on the folding stability. Lysis buffer enhances the stabilizing effect of Ficoll on PGK and inhibits the stabilizing effect of Ficoll on VlsE. We demonstrate that a combination of 150 mg/ml Ficoll and 60% lysis buffer can be used as an in vitro mimic to account for both crowding and non-steric effects on PGK and VlsE stability and folding kinetics in the cell. Our results also suggest that this mixture is close to the point where phase separation will occur. The simple mixture proposed here, based on commercially available reagents, could be a useful tool to study a variety of cytoplasmic protein interactions, such as folding, binding and assembly, and enzymatic reactions. SIGNIFICANCE STATEMENT: The complexity of the in-cell environment is difficult to reproduce in the test tube. Here we validate a mimic of cellular crowding and sticking interactions in a test tube using two proteins that are differently impacted by the cell: one is stabilized and the other is destabilized. This mimic is a starting point to reproduce cellular effects on a variety of protein and biomolecular interactions, such as folding and binding.

SUBMITTER: Davis CM

PROVIDER: S-EPMC7096716 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Empirical binding models have previously been investigated for the energetics of protein complexation (DeltaG models) and for the influence of mutations on complexation (i.e. differences between wild-type and mutant complexes, DeltaDeltaG models). We construct binding models to directly compare these processes, which have generally been studied separately. RESULTS: Although reasonable fit models were found for both DeltaG and DeltaDeltaG cases, they differ substantially. In a dataset curated for the absence of mainchain rearrangement upon binding, non-polar area burial is a major determinant of DeltaG models. However this DeltaG model does not fit well to the data for binding differences upon mutation. Burial of non-polar area is weighted down in fitting of DeltaDeltaG models. These calculations were made with no repacking of sidechains upon complexation, and only minimal packing upon mutation. We investigated the consequences of more extensive packing changes with a modified mean-field packing scheme. Rather than emphasising solvent exposure with relatively extended sidechains, rotamers are selected that exhibit maximal packing with protein. This provides solvent accessible areas for proteins that are much closer to those of experimental structures than the more extended sidechain regime. The new packing scheme increases changes in non-polar burial for mutants compared to wild-type proteins, but does not substantially improve agreement between DeltaG and DeltaDeltaG binding models. CONCLUSION: We conclude that solvent accessible area, based on modelled mutant structures, is a poor correlate for DeltaDeltaG upon mutation. A simple volume-based, rather than solvent accessibility-based, model is constructed for DeltaG and DeltaDeltaG systems. This shows a more consistent behaviour. We discuss the efficacy of volume, as opposed to area, approaches to describe the energetic consequences of mutations at interfaces. This knowledge can be used to develop simple computational screens for binding in comparative modelled interfaces.

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An in vitro mimic of in-cell solvation for protein folding studies.

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