Glucosylation of beta-lactoglobulin lowers the heat capacity change of unfolding; a unique way to affect protein thermodynamics

Abstract

Chemical glycosylation of proteins occurs in vivo spontaneously, especially under stress conditions, and has been linked in a number of cases to diseases related to protein denaturation and aggregation. It is the aim of this work to study the origin of the change in thermodynamic properties due to glucosylation of the folded beta-lactoglobulin A. Under mild conditions Maillard products can be formed by reaction of epsilon-amino groups of lysines with the reducing group of, in this case, glucose. The formed conjugates described here have an average degree of glycosylation of 82%. No impact of the glucosylation on the protein structure is detected, except that the Stokes radius was increased by approximately 3%. Although at ambient temperatures the change in Gibbs energy of unfolding is reduced by 20%, the denaturation temperature is increased by 5 degrees C. Using a combination of circular dichroism, fluorescence, and calorimetric approaches, it is shown that the change in heat capacity upon denaturation is reduced by 60% due to the glucosylation. Since in the denatured state the Stokes radius of the protein is not significantly smaller for the glucosylated protein, it is suggested that the nonpolar residues associate to the covalently linked sugar moiety in the unfolded state, thereby preventing their solvent exposure. In this way coupling of small reducing sugar moieties to solvent exposed groups of proteins offers an efficient and unique tool to deal with protein stability issues, relevant not only in nature but also for technological applications.

Figure 1.

Mass spectrometric analysis of nonmodified (black line) and glucosylated β-lactoglobulin A (gray line).

Figure 2.

Conformational properties of 0.1 mg/mL nonmodified (black lines) and glucosylated (gray lines) β-lactoglobulin A in 10 mM phosphate- buffer (pH 7.0) at 20°C at a secondary (far-UV CD) (A) and tertiary (tryptophan fluorescence) (B) folding level.

Figure 3.

Evaluation of protein conformational stability. (A) Urea denaturation equilibrium study of nonmodified (black) and glucosylated (gray) β-lactoglobulin A as monitored by the tryptophan fluorescence at 320 nm as a function of the urea concentration at 20°C. The lines represent the fits of the two-state unfolding analysis of the data. (B) The CD intensity at 293 nm in the near-UV CD spectra is monitored as function of temperature for the two proteins. Upon reworking the observed intensities in terms of unfolded fraction and taking the derivative over temperature and subsequent smoothing, the curves presented in this panel are obtained.

Figure 4.

Enthalpy change of nonmodified (black symbols) and glucosylated (gray symbols) β-lactoglobulin A as a function of the denaturation temperature. Data are obtained by fitting temperature traces of near-UV CD intensities at 293 for various denaturant concentrations (0–3.2 M urea).

Figure 5.

(A) Differential scanning calorimetric profile of 20 mg/mL nonmodified (black line) and glucosylated (gray line) β-lactoglobulin A (b-LGA) in 10 mM phosphate-buffer (pH 7.0). The curves are displaced vertically for clarity. The dashed lines represent the baselines before and after denaturation. (B) Plot of the enthalpy change vs. the denaturation temperature for nonmodified (black symbols) and glucosylated (gray symbols) b-LGA as obtained using DSC (pH 2.0) and a varying phosphate concentration ranging from 0–200 mM.

Figure 6.

Diffusion time of nonmodified β-lactoglobulin A in the presence of 0 and 8 M urea, as monitored by fluorescence correlation spectroscopy.

Figure 7.

Change in Gibbs energy as a function of temperature. The dashed lines (black for nonmodified, gray for glucosylated b-LGA) are the established curves according to the method of Greene and Pace (1974) using data obtained by DSC (ΔH for nonmodified and glucosylated β-lactoglobulin A [b-LGA] are 380 and 340 kJ/mol, with corresponding T_ds of 346 and 351 K, respectively). Values for ΔCp (11.0 and 4.5 kJ/mol for nonmodified and glucosylated b-LGA, respectively, are established as described in this work). The symbols reflect experimental values for nonmodified (black) and glucosylated (gray) b-LGA as derived from equilibrium denaturant unfolding studies carried out at different temperatures.