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Articles

Locating the binding sites of retinol and retinoic acid with milk β-lactoglobulin

, , , , &
Pages 437-447 | Received 08 Jan 2012, Accepted 25 Feb 2012, Published online: 11 Jun 2012
 

Abstract

β-lactoglobulin (β-LG) is a member of lipocalin superfamily of transporters for small hydrophobic molecules such as retinoids. We located the binding sites of retinol and retinoic acid on β-LG in aqueous solution at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods, and molecular modeling. The retinoid-binding sites and the binding constants as well as the effect of retinol and retinoic acid complexation on protein stability and secondary structure were determined. Structural analysis showed that retinoids bind strongly to β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K retinol- β -LG = 6.4 (± .6) × 106 M−1 and K retinoic acid- β -LG = 3.3 (± .5) × 106 M−1. The number of retinoid molecules bound per protein (n) is 1.1 (± .2) for retinol and 1.5 (± .3) for retinoic acid. Molecular modeling showed the participation of several amino acids in the retinoid–protein complexes with the free binding energy of −8.11 kcal/mol for retinol and −7.62 kcal/mol for retinoic acid. Protein conformation was altered with reduction of β-sheet from 59 (free protein) to 52–51% and a major increase in turn structure from 13 (free protein) to 24–22%, in the retinoid–β-LG complexes, indicating a partial protein destabilization.

Acknowledgments

The financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC) is highly appreciated.

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