Residue mutations in the sweetness loops for the sweet-tasting protein brazzein

Abstract

To identify critical residues, important for sweetness, of the sweet protein brazzein, 11 mutants of the residues in three loops of brazzein were constructed by site-directed mutagenesis. We found that mutations of Glu41 to Ala, Lys, or Arg at position 41 in loop 40-43 made the molecules significantly sweeter than brazzein, while mutations at two distant residues (changing Arg43 to Lys or Glu) decreased sweetness. A similar pattern occurred at loop 30-33. where mutation of the His31 to Arg significantly increased sweetness, while mutations at positions 30 or 33 in the immediate vicinity of this region significantly decreased sweetness. In addition, a Gln17 residue in the loop 9-19 was necessary for structural integrity. From these results, we suggest that the loops containing His31 and Glu41 are critical regions of the molecule for eliciting sweetness, and the charge and/or structure of the side chain of these residues play an important role in the multi-point interactions between brazzein and the sweet-taste receptor. (C) 2011 Elsevier Ltd. All rights reserved.