Probing the sweet determinants of brazzein: wild-type brazzein and a tasteless variant, brazzein-ins(R18a-I18b), exhibit different pH-dependent NMR chemical shifts.
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 Leu18 and Ala19 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 alpha-helix, and around the Cys16-Cys37 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 (His31, Arg33, Glu41, Arg43, Asp50, and Tyr54) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His31, Glu41, Asp50, and Tyr54 were unaltered by the insertion. The insertion led to large chemical shift and pKa perturbation of Glu36, 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).
Zhao
Qin
Q
Department of Biochemistry, University of Wisconsin Madison, College of Agricultural and Life Sciences, USA. qin@nmrfam.wisc.edu
Song
Jikui
J
Jin
Zheyuan
Z
Danilova
Vicktoria
V
Hellekant
Göran
G
Markley
John L
JL
D014644:Genetic Variation / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics*; D017434:Protein Structure, Tertiary; D013549:Sweetening Agents / Q000737:chemistry*
D010940:Plant Proteins; D013549:Sweetening Agents; C090521:brazzein protein, Pentadiplandra brazzeana