Probing the sweet determinants of brazzein: Wild-type brazzein and a tasteless variant, brazzein-ins(R18a-I18b), exhibit different pH-dependent NMR chemical shifts

Qin Zhao, Jikui Song, Zheyuan Jin, Vicktoria Danilova, Göran Hellekant, John L. Markley

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Abstract

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 α-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 α-helix and the second β-strand. In particular, several residues of brazzein previously shown to be essential for its sweetness (His31, Arg33, Glu41, Arg 43, 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).

Original languageEnglish (US)
Pages (from-to)256-263
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume335
Issue number1
DOIs
StatePublished - Sep 16 2005

Bibliographical note

Funding Information:
This work was supported by NIH Grants DC006016 (G.H.) and RR02301 (J.L.M.). Chemical shifts and pK a values described here have been deposited at BioMagResBank (BMRB 5295 for brazzein and BMRB 5296 for brazzein-ins(R 18a -I 18b )).

Funding Information:
We thank Dr. Ed Mooberry for assistance with NMR hardware and Dr. Fariba Assadi-Porter for providing the plasmid coding for wild-type brazzein. This work was supported by NIH Grants R01 DC006016 (G.H., P.I.) and P41 RR02301 (J.L.M., P.I.), which provides partial support for the National Magnetic Resonance Facility at Madison.

Keywords

  • Brazzein
  • Mutagenesis
  • NMR spectroscopy
  • Sweet-taste determinants
  • pK

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