Brazzein is a small, intensely sweet protein. As a probe of the functional properties of its solvent-exposed loop, two residues (Arg-Ile) were inserted between Leu(18) and Ala(19) of brazzein. Psychophysical testing demonstrated that this mutant is totally tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that residues affected by the insertion are localized to the mutated loop, the region of the single a-helix, and around the Cys(16)-Cys(37) disulfide bond. Residues unaffected by this mutation included those near the C-terminus and in the loop connecting the alpha-helix and the second beta-strand. In particular, several residues of brazzein previously shown to be essential for its sweetness (His(31), Arg(33), Glu(41), Arg(43), Asp(50), and Tyr(54)) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His(31), Glu(41), Asp(50), and Tyr(54) were unaltered by the insertion. The insertion led to large chemical shift and pK(a) perturbation of Glu(36), a residue shown previously to be important for brazzein's sweetness. These results serve to refine the known sweetness determinants of brazzein and lend further support to the idea that the protein interacts with a sweet-taste receptor through a multi-site interaction mechanism, as has been postulated for brazzein and other sweet proteins (monellin and thaumatin). (c) 2005 Elsevier Inc. All rights reserved.