Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu

Abstract

The natural cellular milieu is crowded by large quantities of various biological macromolecules. This complex environment is characterized by a limited amount of unoccupied space, limited amounts of free water, and changed solvent properties. Obviously, such a tightly packed cellular environment is poorly mimicked by traditional physiological conditions, where low concentrations of a protein of interest are analyzed in slightly salted aqueous solutions. An alternative is given by the use of a model crowded milieu, where a protein of interest is immersed in a solution containing high concentrations of various polymers that serve as model crowding agents. An expected outcome of the presence of such macromolecular crowding agents is their ability to increase conformational stability of a globular protein due to the excluded volume effects. In line with this hypothesis, the behavior of a query protein should be affected by the hydrodynamic size and concentration of an inert crowder (i.e., an agent that does not interact with the protein), whereas the chemical nature of a macromolecular crowder should not play a role in its ability to modulate conformational properties. In this study, the effects of different crowding agents (polyethylene glycols (PEGs) of various molecular masses (PEG-600, PEG-8000, and PEG-12000), Dextran-70, and Ficoll-70) on the spectral properties and unfolding–refolding processes of the super-folder green fluorescent protein (sfGFP) were investigated. sfGFP is differently affected by different crowders, suggesting that, in addition to the expected excluded volume effects, there are some changes in the solvent properties.

Nanoparticle-Mediated Assembly of Peptoid Nanosheets Functionalized with Solid-Binding Proteins: Designing Heterostructures for Hierarchy

“…Line scans performed on high-resolution AFM images revealed two different types of features: fairly sharp spots about 6.0 ± 1.4 nm in width and 1.6 ± 0.35 nm in height (Figures B and S9) and larger spots of variable width and lower height (1.1 ± 0.1 nm; Figure S10). The former features are consistent with the crystallographic dimensions (4.2 × 2.4 nm 2 ) and the Stokes radius (2.4 nm) of sfGFP molecules lying on their side, perhaps as a result of the drying process. We assign the lower height and irregular spots to proteins that have become deformed or misfolded during sample preparation and/or upon drying (unfolded sfGFP has a Stokes radius of 4.8 nm) .…”

“…The former features are consistent with the crystallographic dimensions (4.2 × 2.4 nm 2 ) and the Stokes radius (2.4 nm) of sfGFP molecules lying on their side, perhaps as a result of the drying process. We assign the lower height and irregular spots to proteins that have become deformed or misfolded during sample preparation and/or upon drying (unfolded sfGFP has a Stokes radius of 4.8 nm) . The underlying protein layer appears to be primarily composed of such deformed or misfolded polypeptides because the nanosheet top deck experienced only a 1.0 ± 0.1 nm height gain relative to unmodified nanosheets (compare the line scans in Figure A,B).…”

In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria

“…GFP levels were rather associated with stability of mRNA folding near the ribosomal binding site, mRNA levels and mRNA degradation patterns [ 60 ]. Other factors that might also affect sfGFP expression could be associated with mRNA:ncRNA interactions, tRNA abundance, co-translational folding, the translation rhythm, speed of folding and the macromolecular crowding [ 61 – 68 ].…”

β-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis

“…In such states, monomers of the β-barrel proteins can interact with one another to form amyloid fibrils. Conditions initiating the fibrillogenesis of β-barrel proteins in vitro [ 18 , 186 , 216 ] suggest that the aggregation of these proteins in vivo can be promoted by some stressful external influences destabilizing the native protein structure, as well as specific cellular cofactors. Additionally, the conditions of macromolecular crowding—i.e., limited available space due to the high total concentration of macromolecules inside the cell—in some cases, are likely to stimulate the aggregation of β-barrel proteins [ 18 ].…”