Structure and Conformational Properties of d-Glucose/d-Galactose-Binding Protein in Crowded Milieu

Abstract

Conformational changes of d-glucose/d-galactose-binding protein (GGBP) were studied under molecular crowding conditions modeled by concentrated solutions of polyethylene glycols (PEG-12000, PEG-4000, and PEG-600), Ficoll-70, and Dextran-70, addition of which induced noticeable structural changes in the GGBP molecule. All PEGs promoted compaction of GGBP and lead to the increase in ordering of its structure. Concentrated solutions of PEG-12000 and PEG-4000 caused GGBP aggregation. Although Ficoll-70 and Dextran-70 also promoted increase in the GGBP ordering, the structural outputs were different for different crowders. For example, in comparison with the GGBP in buffer, the intrinsic fluorescence spectrum of this protein was shifted to short-wave region in the presence of PEGs but was red-shifted in the presence of Ficoll-70 and Dextran-70. It was hypothesized that this difference could be due to the specific interaction of GGBP with the sugar-based polymers (Ficoll-70 and Dextran-70), indicating that protein can adopt different conformations in solutions containing molecular crowders of different chemical nature. It was also shown that all tested crowding agents were able to stabilize GGBP structure shifting the GGBP guanidine hydrochloride (GdnHCl)-induced unfolding curves to higher denaturant concentrations, but their stabilization capabilities did not depend on the hydrodynamic dimensions of the polymers molecules. Refolding of GGBP was complicated by protein aggregation in all tested solutions of crowding agents. The lowest yield of refolded protein was achieved in the highly concentrated solutions of PEG-12000. These data support the previous notion that the influence of macromolecular crowders on proteins is rather complex phenomenon that extends beyond the excluded volume effects.

Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

“…This indicates that the volume expansion of the sensing protein alone is not much affected by this crowding agent. Interestingly, our observation somehow disagrees with recent findings which claim some PEG-induced compaction of the glucose binding protein [28]. Most probably this disagreement is caused by the rather different experimental parameters which were measured in both studies.…”

Cascade Kinetics in an Enzyme-Loaded Aqueous Two-Phase System

“…It is evident that the upper ATPS phase has one order of magnitude lower V max , although, all K m are similar. Thus, the observed behavior could be explained by the ability of the PEG polymer to interfere with hydrophobic interactions inside the protein structure and partially denature enzymes, as previously reported for some specific interaction or crowding agents with substrates. , …”

“…Thus, the observed behavior could be explained by the ability of the PEG polymer to interfere with hydrophobic interactions inside the protein structure and partially denature enzymes, as previously reported for some specific interaction or crowding agents with substrates. 5,52 Scheme 2. Two Cascade Reactions Investigated in the Current Study a a Both of them starting from the oxidation of uric acid, which is followed by subsequent formation of the colored product from either ABTS or guaiacol oxidation.…”

Photophysical Properties of BADAN Revealed in the Study of GGBP Structural Transitions

“…These results may be due to the fact that the microenvironment of BADAN and GGBP tryptophan residues reflects the conformational changes in different regions of the protein. BADAN is localized between the GGBP domains in the active center of the protein, while four out of five GGBP tryptophan residues are located in the C-terminal domain of GGBP [38][39][40]. However, there is still no exhaustive explanation of this effect.…”