Brazzein is a small (6.5 kDa) sweet-tasting protein originating from the fruit of Pentadiplandra brazzeana, a plant found in West Africa. Brazzein like all classes of sweet compounds is perceived through the activation of the T1R2/T1R3 heterodimeric sweet-taste receptor. T1R2 and T1R3 subunits are members of the small family of class C G-protein coupled receptors (GPCRs). Class C GPCRs possess a large N-terminal domain (NTD) linked to seven transmembrane domain by a cysteine rich domain (CRD). The NTD of T1R2 (T1R2-NTD) has been shown to contain the primary binding site for most of the sweet ligands. However, brazzein has been shown to require CRD of human T1R3 for receptor activation [1]. In contrast, molecular modeling and docking studies have proposed that brazzein may interact primarily with T1R2-NTD and makes also favorable contacts with T1R3-NTD [2]. To elucidate the contribution of T1R2-NTD to brazzein detection, we recombinantly expressed T1R2-NTD in Escherichia coli as a soluble cytoplasmic protein. Human T1R2-NTD was purified using three chromatography steps and characterized for its ability to interact with recombinant brazzein secreted by the yeast Pichia pastoris [3]. Brazzein/T1R2-NTD interactions were measured using Bio-Layer Interferometry (BLI). This optical technique analyzes variations in the interference pattern generated from visible light reflected from an optical layer and a biolayer containing immobilized protein of interest. This recent method is powerful for studying protein-protein interactions and measuring both affinity constants and kinetic parameters. BLI experiments were performed first by immobilizing T1R2-NTD onto the biosensor and measuring brazzein binding. In a second experiment, brazzein was immobilized onto the biosensor and T1R2-NTD binding was followed using BLI. Both experiments demonstrated that T1R2-NTD binds brazzein with a Kd value of approximately 30 µM. This affinity is in agreement with the capacity of brazzein to activate T1R2/T1R3 receptor heterologously expressed in HEK cells and with sensory experiments conducted on humans [3]. These data suggest that T1R2-NTD is the primary site for brazzein binding, which will be further investigated by site-directed mutagenesis conducted on brazzein and T1R2-NTD. 1. Jiang P, Ji Q, Liu Z, et al. The cysteine-rich region of T1R3 determines responses to intensely sweet proteins. The Journal of biological chemistry. 2004;279(43):45068-75. 2. Walters DE, Hellekant G. Interactions of the sweet protein brazzein with the sweet taste receptor. Journal of agricultural and food chemistry. 2006;54(26):10129-33. 3. Poirier N, Roudnitzky N, Brockhoff A, et al. Efficient production and characterization of the sweet-tasting brazzein secreted by the yeast Pichia pastoris. Journal of agricultural and food chemistry. 2012;60(39):9807-14.