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.

Keywords: ">d-galactose-binding protein; ">d-glucose/; circular dichroism; intrinsic fluorescence; macromolecular crowding; polymers; protein aggregation; protein folding; protein unfolding.

Figure 1

Characteristics of the GGBP intrinsic fluorescence in the presence of different crowding agents. The excitation wavelength was 297 nm.

Figure 2

Far-UV CD spectra of GGBP in the presence of PEG of different molecular weight. (A): PEG-12000; (B): PEG-4000; (C): PEG-600.

Figure 3

Far-UV CD spectra of GGBP in the presence of Ficoll-70 (A) and Dextran-70 (B).

Figure 4

Unfolding/refolding transitions of GGBP induced by GdnHCl in the presence of different concentrations of PEGs of various molecular weights recorded as the GdnHCl dependencies of parameter A = I₃₂₀/I₃₆₅. The excitation wavelength was 297 nm. The unfolding of GGBP is represented by open symbols, whereas refolding is shown by closed symbols. The black curve is equilibrium GdnHCl-induced unfolding of GGBP in the absence of crowding agents. The gray curve in the upper left panel represents the equilibrium GdnHCl-induced unfolding transition of the glucose bound GGBP. The gray dashed curve in the same plot shows the equilibrium GdnHCl-induced unfolding of the GGBP sample prepared by the pre-incubation in 2.5 M GdnHCl solution followed by the extensive dialysis.

Figure 5

Unfolding/refolding transitions of GGBP in the presence of different concentrations of Ficoll-70 and Dextran-70 recorded by the dependence of parameter A = I₃₂₀/I₃₆₅ on GdnHCl concentration. The excitation wavelength was 297 nm. The designations as in Figure 4.

Figure 6

Unfolding/refolding transitions of GGBP in the presence of different concentrations of PEGs with different molecular weight recorded as the dependence of the intrinsic fluorescence intensity of GGBP on GdnHCl concentration. The excitation wavelength was 297 nm, the emission wavelength was 365 nm. Other designations as in Figure 4.

Figure 7

Unfolding/refolding transitions of GGBP in the presence of different concentrations of Ficoll-70 and Dextran-70 recorded as the dependence of the intrinsic fluorescence intensity of GGBP on GdnHCl concentration. The excitation wavelength was 297 nm, emission wavelength was 365 nm. Other designations as in Figure 4.

Figure 8

Unfolding/refolding transitions of GGBP in the presence of different concentrations of PEG of different molecular weight recorded by the dependence of the anisotropy of intrinsic fluorescence of GGBP on GdnHCl concentration. The excitation and emission wavelengths were 297 and 365 nm, respectively. Other designations as in Figure 4.

Figure 9

Unfolding/refolding transitions of GGBP in the presence of different concentrations of Ficoll-70 and Dextran-70 recorded as the dependence of the anisotropy of intrinsic fluorescence on GdnHCl concentration. The excitation wavelength was 297 nm, the emission wavelength was 365 nm. Other designations as in Figure 4.

Figure 10

Unfolding/refolding transitions of GGBP in the presence of different concentrations of PEGs with different molecular weight recorded as dependence of GGBP molar ellipticity at 222 nm on GdnHCl concentration. The designations as in Figure 4.

Figure 11

Unfolding/refolding transitions of GGBP in the presence of different concentrations of Ficoll-70 and Dextran-70 recorded by the dependence of the molar ellipticity at 222 nm on GdnHCl concentration. Other designations as in Figure 4.