Osmolyte-Like Stabilizing Effects of Low GdnHCl Concentrations on d-Glucose/d-Galactose-Binding Protein.

Alexander V Fonin,Irina M Kuznetsova,Maria Staiano,Alexandra D Golikova,Irina A Zvereva,Sabato D'Auria,Vladimir N Uversky,Konstantin K Turoverov

doi:10.3390/ijms18092008

Abstract

The ability of d-glucose/d-galactose-binding protein (GGBP) to reversibly interact with its ligands, glucose and galactose, makes this protein an attractive candidate for sensing elements of glucose biosensors. This potential is largely responsible for attracting researchers to study the conformational properties of this protein. Previously, we showed that an increase in the fluorescence intensity of the fluorescent dye 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN) is linked to the holo-form of the GGBP/H152C mutant in solutions containing sub-denaturing concentrations of guanidine hydrochloride (GdnHCl). It was hypothesized that low GdnHCl concentrations might lead to compaction of the protein, thereby facilitating ligand binding. In this work, we utilize BADAN fluorescence spectroscopy, intrinsic protein UV fluorescence spectroscopy, and isothermal titration calorimetry (ITC) to show that the sub-denaturing GdnHCl concentrations possess osmolyte-like stabilizing effects on the structural dynamics, conformational stability, and functional activity of GGBP/H152C and the wild type of this protein (wtGGBP). Our data are consistent with the model where low GdnHCl concentrations promote a shift in the dynamic distribution of the protein molecules toward a conformational ensemble enriched in molecules with a tighter structure and a more closed conformation. This promotes the increase in the configurational complementarity between the protein and glucose molecules that leads to the increase in glucose affinity in both GGBP/H152C and wtGGBP.

Highlights

Interactions of proteins with low-molecular weight ligands are crucial for the functioning of living systems
Our data are consistent with the model where low guanidine hydrochloride (GdnHCl) concentrations promote a shift in the dynamic distribution of the protein molecules toward a conformational ensemble enriched in molecules with a tighter structure and a more closed conformation
The fluorescence intensity of BADAN linked to the holo-form of glucose/D-galactose-binding protein (GGBP)/H152C first significantly increases in the range of 0.0–0.2 M GdnHCl, remains unchanged in the range of 0.2–1.0 M GdnHCl, and dramatically decreases in the range of 1.0–1.5 M GdnHCl in parallel with the protein unfolding

Summary

Introduction

Interactions of proteins with low-molecular weight ligands are crucial for the functioning of living systems. The conformational selection model suggests the existence in apo-form of an ensemble of protein conformers with different ligand binding affinities [2,3,4]. These conformers have comparable free energies [5], and ligands predominantly interact with a conformer, whose active center is the most complementary to the structure of the ligand [6]. This ensures that such an interaction is the most advantageous thermodynamically According to this model, ligand binding to protein does not induce structural changes of the protein molecule, but causes the redistribution of the ensemble of protein conformers in solution. The interaction of some proteins with their ligands was described by a mixed mechanism, including elements of both conformational selection and induced fit models [8,9,10]

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