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 α-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 α-helix and the second β-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).