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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 StatementThe 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.
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Intensity correlation analysis of Raman spectra of concentrated Ficoll solutions
The intracellular environment is crowded with diverse biomacrolecules (~80-400 mg/ml), likely affecting various biological processes such as protein folding, binding of small molecules, enzymatic activity, and pathological protein aggregation. As a model we have been using solutions of Ficoll, a highly branched polysaccharide, to mimic the environment. Besides its biomedical applications (e.g. blood separation), it has been used as a macromolecular crowder in studies of protein folding and stability, cell volume signaling, tissue engineering, and nanotransport. In this study, our goal is to identify and assess Raman spectral signatures associated with Ficoll molecules and Ficoll-Ficoll interactions for future investigations of crowding effects. In addition to the Raman peaks of water (~1640 cm-1 and ~3200 cm-1) and dissolved O2 (~1556 cm-1) and N2 (~2331 cm-1) we identified a distinct Raman peak (~2900 cm-1) in the 1500-3500 cm-1 wavenumber range, which is associated with Ficoll and CH and CH2 stretching modes. As the Ficoll concentration increases, the intensity of the Ficoll Raman peaks increases while the intensity of the water Raman peaks decreases, the latter likely due to reduction of water content. Further, we have applied the intensity correlation analysis (ICA) method to assess systematic changes of Raman spectra with Ficoll concentration (up to 1000 mg/ml). ICA indicates an overall linear trend over the full wavenumber range, but also shows closed loops that can be attributed to slight changes of the profiles of certain peaks. The results demonstrate ICA as a potential insightful tool for identifying Ficoll in chemical analysis of crowded biological samples.
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- Award ID(s):
- 1955664
- PAR ID:
- 10447138
- Editor(s):
- Maitland, Kristen C.; Roblyer, Darren M.; Campagnola, Paul J.
- Date Published:
- Journal Name:
- Proc. SPIE 11944, Multiscale Imaging and Spectroscopy III
- Page Range / eLocation ID:
- 28
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1117/12.2609947
