Monkey Electrophysiological and Human Psychophysical Responses to Mutants of the Sweet Protein Brazzein: Delineating Brazzein Sweetness

Abstract

Responses to brazzein, 25 brazzein mutants and two forms of monellin were studied in two types of experiments: electrophysiological recordings from chorda tympani S fibers of the rhesus monkey, Macaca mulatta, and psychophysical experiments. We found that different mutations at position 29 (changing Asp29 to Ala, Lys or Asn) made the molecule significantly sweeter than brazzein, while mutations at positions 30 or 33 (Lys30Asp or Arg33Ala) removed all sweetness. The same pattern occurred again at the beta-turn region, where Glu41Lys gave the highest sweetness score among the mutants tested, whereas a mutation two residues distant (Arg43Ala) abolished the sweetness. The effects of charge and side chain size were examined at two locations, namely positions 29 and 36. The findings indicate that charge is important for eliciting sweetness, whereas the length of the side-chain plays a lesser role. We also found that the N- and C-termini are important for the sweetness of brazzein. The close correlation (r = 0.78) between the results of the above two methods corroborates our hypothesis that S fibers convey sweet taste in primates.

Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2–T1R3 Human Sweet Receptor

“…A three-dimensional representation of the protein surface from a high-resolution (RMSD = 0.38 Å) NMR structure of recombinant wild-type brazzein (C. C. Cornilescu, F. M. Assadi-Porter, et al, manuscript in prep. ), provides a visual summary of the taste properties of these variants in the context of those previously reported 34; 35; 36 (Fig. 4).…”

“…In contrast, substitutions in Loop9–19 reduced sweetness by factors of 2–4 34. Substitutions of residue E36 (which lies near the C-terminus in the 3D structure – Site 2) by A, Q, or K significantly reduced sweetness 34; 35; 36. Deletion of Y54 (the C-terminal residue – Site 2) or insertions of two amino acids at the C-terminus (Site 2) completely eliminated sweetness, indicating that the length of the C-terminus is important for brazzein’s sweetness and suggesting that the C-terminus may interact directly with the sweet receptor 34.…”

“…In addition, nearby Loop33 and Loop9–19 (Site 3) make significant contributions to brazzein’s sweetness. Ala-scanning studies aimed at elucidating the physicochemical basis of brazzein’s sweetness 34; 35; 36 demonstrated the importance of charged Arg and Lys residues in the loop regions. Mutation R43A (Site 1) resulted in profoundly reduced activity 34.…”

Sense of Taste in a New World Monkey, the Common Marmoset. II. Link Between Behavior and Nerve Activity

“…We demonstrated this in hamster (Danilova et al 1998b). We also tested this hypothesis in a recent study using S fiber responses as a measure of sweetness (Jin et al 2003). We found a high correlation (0.76) between the S fiber responses to 28 sweeteners in rhesus monkeys and psychophysical results in humans for the same array of compounds.…”

The Proteins of Plant Defensin Family and their Application Beyond Plant Disease Control

“…The solution structure of brazzein has been resolved by nuclear magnetic resonance in 1998 [22]. Following the structure resolved, a series of studies based on the structure are carried out to probe the key elements on the sweet taste of brazzein [23][24][25][26]. These studies recruited the new developed computing simulation tools to predict the key residues on brazzein and one derivate of brazzein displaying higher sweet taste than the naturally produced brazzein is discovered.…”