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

¹ Wageningen Centre for Food Sciences, 6700 AN Wageningen, The Netherlands
² Wageningen University and Research Centre, Wageningen, The Netherlands
³ TNO Nutrition and Food Research, Zeist, The Netherlands

(RECEIVED February 10, 2005; FINAL REVISION April 19, 2005; ACCEPTED April 27, 2005)

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 -lactoglobulin A. Under mild conditions Maillard products can be formed by reaction of -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 ~3%. Although at ambient temperatures the change in Gibbs energy of unfolding is reduced by 20%, the denaturation temperature is increased by 5°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.

Keywords: -lactoglobulin; Maillard reaction; protein stability; heat capacity; glycosylation; thermodynamics

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051405005.

Reprint requests to: Harmen H.J. de Jongh, Wageningen Centre for Food Sciences, Diedenweg 20, 6700 AN Wageningen, The Netherlands; e-mail: harmen.dejongh{at}wur.nl; fax: +31-317-484893.

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