Temperature-dependent conformational change affecting Tyr11 and sweetness loops of brazzein

Abstract

The sweet protein brazzein, a member of the Csβα fold family, contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Nevertheless, a variable temperature study has revealed that the protein undergoes a local, reversible conformational change between 37 and 3°C with a midpoint about 27°C that changes the orientations and side-chain hydrogen bond partners of Tyr8 and Tyr11. To test the functional significance of this effect, we used NMR saturation transfer to investigate the interaction between brazzein and the amino terminal domain of the sweet receptor subunit T1R2; the results showed a stronger interaction at 7°C than at 37°C. Thus the low temperature conformation, which alters the orientations of two loops known to be critical for the sweetness of brazzein, may represent the bound state of brazzein in the complex with the human sweet receptor.

Figure 1

Sections of aromatic ¹³C-HSQC NMR spectra of brazzein containing signals from Tyr11 collected at the temperatures indicated. (Upper row) Cross hairs follow the Tyr11 ¹³C^δ1–¹H^δ1 and ¹³C^δ2–¹H^δ2 signals, which yielded single averaged cross peaks as the result of rapid ring flips. The cross peak broadened below the noise level in spectra taken between 32 and 22°C. (Lower row) Cross hairs follow ¹³C^ε1–¹H^ε1 and ¹³C^ε2–¹H^ε2 signals. In the high-temperature conformation, the ¹³C^ε1–¹H^ε1 and ¹³C^ε2–¹H^ε2 signals yielded a single averaged cross peak as the result of rapid ring flips. The signal broadened beyond detection at 32°C, but appeared as two separate peaks at 27°C and below, as indicative of slow ring flips.

Figure 2

Strips from aromatic 3D ¹³C-NOESY spectra comparing NOE contacts of the Tyr11 ¹H^δ and ¹H^ε protons at (left panels) 37°C and (right panels) 3°C. Unlabeled peaks are either diagonal peaks or belong to other residues.

Figure 3

Comparison of the structures of brazzein determined at (left panels) 37°C and (right panels) 3°C with Tyr8 in gold and Tyr11 in green. Shown are relevant H-bonds (blue dotted lines) and the side chains of residues that significantly change conformation and/or H-bond partners (Glu9, Pro12, and Lys42). Disulfide bonds are represented in yellow. (A) Ribbon diagrams of the lowest energy structures of brazzein. (B) Detail showing the H-bond partners of Tyr 8 and Tyr11.

Figure 4

Comparison of the environments of Tyr8 in gold and Tyr11 in green in the brazzein structures at (left panel) 37°C and (right panel) 3°C.

Figure 5

Top: STD spectra of ATD-T1R2 preparations containing brazzein collected at temperatures 37, 27, and 7°C. STD signals of brazzein in the methyl and amide/aromatic regions appearing at 7°C indicate binding to the receptor. Bottom: 1D ¹H NMR control spectrum of brazzein. Peaks labeled with an asterisk arise from detergent binding to the receptor. Slight changes in peak positions are the result of temperature differences.