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

Alexander Fonin,Irina Kuznetsova,Konstantin Turoverov,Asiya Sitdikova,Sergey Silonov,Vladimir Uversky

doi:10.3390/molecules22020244

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.

Highlights

Protein folding has been intensively studied for the past 50 years
The values of Stern-Volmer constant measured for GGBPtryptophan in the presence of crowders are similar to that recorded protein in the presence glucose. for of protein residues in buffer solution
In one of our papers [19] we have shown that the complexation of glucose/D-galactose-binding protein (GGBP) with glucose causes a shift in the middle of the transition between the native and unfolded states of protein from 0.4 M guanidine hydrochloride (GdnHCl) to 0.9 M GdnHCl

Summary

Introduction

Most of the data related to folding of different proteins were obtained as a result of in vitro experiments. Molecules 2017, 22, 244 these experiments were performed in dilute solutions, but the processes of in vitro and in vivo folding are significantly different [1]. Protein folding in the cell is intimately associated with protein biosynthesis. When a nascent polypeptide chain leaves the ribosome, the protein begins to fold immediately, giving rise to the cotranslational folding process. Chaperones and foldases (i.e., enzymes catalyzing cis-trans isomerization of proline residues and formation of ‘correct’ disulfide bonds) are required for correct protein folding in the cellular environment [1]. Protein folding in vivo occurs in highly crowded environment; i.e., under conditions of minimal free space and permanent steric contacts of a protein undergoing folding process with other macromolecules

Objectives

Methods

Results

Conclusion