Structure Of Thaumatin Research Articles

Structure of thaumatin under acidic conditions: Structural insight into the conformations in lysine residues responsible for maintaining the sweetness after heat-treatment

Thaumatin is an intensely sweet-tasting protein. Its sweetness persists when heated under acidic conditions, but disappears when heated at a pH above 7.0. To clarify how the structural features of thaumatin resist insoluble aggregation during heating under acidic conditions, we analysed its crystal structure obtained at pH 4.0, 6.0, and 8.0. Simultaneously, the melting temperature (Tm) at these pH levels was determined using differential scanning fluorimetry. At pH 4.0, the Tm of thaumatin was substantially lower and the overall B-factor value of its structure was higher than those at pH 6.0. Interestingly, the relative B-factor values for most lysine residues decreased as the pH reduced. These results suggest that the overall structure at pH 4.0 becomes flexible but the relative flexibility of some regions is lower than that at pH 6.0. Thus, the reduction in relative flexibility might play an important role in preventing thermal aggregation, thereby maintaining the sweetness.

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50nM. Although the sweetness remains when thaumatin is heated at 80°C for 4h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0Å. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of a Cα atom to be substantially greater in the large disulfide-rich region of domain II, especially residues 154–164, suggesting that a loop region in domain II to be affected by solvent conditions. Furthermore, B-factors of Lys137, Lys163, and Lys187 were significantly affected by pH change, suggesting that a striking increase in the mobility of these lysine residues, which could facilitate a reaction with a free sulfhydryl residue produced via the β-elimination of disulfide bonds by heating at a pH above 7.0. The increase in mobility of lysine residues as well as a loop region in domain II might play an important role in the heat-induced aggregation of thaumatin above pH 7.0.

Crystal structure of the sweet-tasting protein thaumatin II at 1.27 Å

Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 Å. Overall structure of thaumatin II is similar to thaumatin I, but a slight shift of the Cα atom of G96 in thaumatin II was observed. Furthermore, the side chain of residue 67 in thaumatin II is highly disordered. Since residue 67 is one of two residues critical to the sweetness of thaumatin, the present results suggested that the critical positive charges at positions 67 and 82 are disordered and the flexibility and fluctuation of these side chains would be suitable for interaction of thaumatin molecules with sweet receptors.

Biochemical and structural characterization of TLXI, the Triticum aestivum L. thaumatin-like xylanase inhibitor

Thaumatin-like xylanase inhibitors (TLXI) are recently discovered wheat proteins. They belong to the family of the thaumatin-like proteins and inhibit glycoside hydrolase family 11 endoxylanases commonly used in different cereal based (bio)technological processes. We here report on the biochemical characterisation of TLXI. Its inhibition activity is temperature- and pH-dependent and shows a maximum at approximately 40°C and pH 5.0. The TLXI structure model, generated with the crystal structure of thaumatin as template, shows the occurrence of five disulfide bridges and three β-sheets. Much as in the structures of other short-chain thaumatin-like proteins, no α-helix is present. The circular dichroism spectrum of TLXI confirms the absence of α-helices and the presence of antiparallel β-sheets. All ten cysteine residues in TLXI are involved in disulfide bridges. TLXI is stable for at least 120 min between pH 1–12 and for at least 2 hours at 100°C, making it much more stable than the other two xylanase inhibitors from wheat, i.e. Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibitor protein (XIP). This high stability can probably be ascribed to the high number of disulfide bridges, much as seen for other thaumatin-like proteins.

Crystallization in the presence of glycerol displaces water molecules in the structure of thaumatin.

The intensely sweet protein thaumatin has been crystallized at 293 K in the presence of sodium tartrate and 25%(v/v) glycerol for X-ray diffraction data collection at 100 K. A comparison of the three-dimensional structure model derived from a crystal grown in the presence of glycerol with that of a control deprived of this additive reveals only minor changes in the overall structure but a approximately 20% reduction in the number of water molecules. X-ray topography analyses show that the overall quality of the crystals prepared in the presence of this cryoprotectant is enhanced.

X-ray diffraction properties of protein crystals prepared in agarose gel under hydrostatic pressure

Crystals of thaumatin and of turkey and hen lysozyme, prepared in a batch at 293 K under hydrostatic pressures in the 0.1–150 MPa range, have been analyzed to investigate how this parameter influences crystallization and if depressurization introduces defects in the crystalline lattice. The X-ray diffraction properties of depressurized crystals have been compared with those of unpressurized control crystals prepared under otherwise identical conditions at atmospheric pressure (0.1 MPa). Independently of pressure, the crystals of each protein belong to the same space group and have cell parameters identical to those of the controls. Their diffraction limit is more dependent upon crystal volume than upon pressure. Crystal mosaicity, expressed as the full-width at half-maximum w of the Bragg reflection profile, was used to quantify the defects in the lattice. While the quality of lysozyme crystals deteriorates as pressure increases, that of tetragonal thaumatin crystals becomes more homogeneous. The first result correlates with the apparition of cleavages oriented perpendicularily to the crystal's c-axis. The second one is interpreted by a gradual selection of well-formed nuclei. The 3D structure of thaumatin in a crystal that was grown under a pressure of 150 MPa and returned to atmospheric pressure was solved. The fold of the polypeptide chain and the distribution of water molecules was compared with those in a reference crystal grown at 0.1 MPa.The results suggest that the crystal lattice is elastic. A possible application of pressure to the preparation of high quality protein crystals is discussed in the light of these results.

Effects of pressure on the crystallization and the solubility of proteins in agarose gel

Crystals of thaumatin and of lysozyme from turkey and hen egg white have been prepared in batch at 20°C under hydrostatic pressures in the 0.1–220 MPa range. The latter model protein served as a reference in this comparative study. Crystallization was performed in an agarose gel in contrast to former studies under pressure that were conducted in solution. After depressurization, the habit, number, length, shape and solubility of crystals were compared to those of control crystals that were prepared at atmospheric pressure (0.1 MPa). For the three proteins, the number of the crystals increases with pressure. For thaumatin and hen lysozyme, crystal length decreases but for turkey lysozyme it increases. While the solubility of the first protein decreases, that of both lysozymes increases. The relationship between solubility and pressure is linear in the three cases. The crystallization volumes Δ V are −11 cm 3 mol −1 for thaumatin, +15 cm 3 mol −1 for turkey lysozyme and +3 cm 3 mol −1 for hen lysozyme. For the lysozymes, they are explained by pressure-dependent conformational changes. The structure of thaumatin appears to be less deformable under pressure.

Crystal structure of a sweet tasting protein thaumatin I, at 1·65 Å resolution

The crystal structure of thaumatin I, a potently sweet protein isolated from the fruits of the West African shrub, Thaumatococcus danielli Benth, has been refined at a resolution better than 1·65 Å using a combination of energy minimization and stereochemically restrained least-squares methods. The final model consists of all 207 amino acids, 28 alternate amino acid conformers and 236 waters, with a crystallographic R-factor of 0·145 for 19,877 reflections having F > 4σ F between 10·0 Å and 1·65 Å ( R = 0.167 for all 24,022 reflections). The model has good stereochemistry, with root-mean-square deviations from ideal values for bond and angle distances of 0·014 Å and 0·029 Å, respectively. 74The estimated root-mean-square co-ordinate error is 0·15 Å. The current model confirms the previously reported 3·1 Å C α trace in both main chain connectivity and disulfide topology, including two disulfide bonds, that differed from the earlier reported biochemical determination. The structure contains three domains. The core of the molecule consists of an eleven-stranded, flattened β-sandwich folded into two Greek key motifs. All β-strands in this sandwich are antiparallel except the parallel N-terminal and the C-terminal strands. The average hydrogen bond length in this sandwich is 2·89 Å, with an angle of 155·1 °. Two β-bulges are found in one of the sheets. The second domain consists of two β-strands forming a β-ribbon and connected by an ω-loop, and contains a proline residue in cis conformation. This structural motif folds back against the main sandwich to form a smaller sandwich-like structure. The third domain is a disulfide-rich region stretching away from the sandwich portion of the molecule. It contains one α-helix and three short helical fragments. Two of the helical segments are connected by an unusually sharp turn, stabilized by a disulfide bridge. One of the three disulfide bonds in this domain takes on two conformations.

Crystal structure of the intensely sweet protein monellin.

Two unusual proteins, discovered in African berries, possess the interesting property of having a very high specificity for the sweet receptors. These proteins, monellin and thaumatin, are approximately 100,000 times sweeter than sugar on a molar basis and several thousand times sweeter on a weight basis. Neither contains carbohydrates or modified amino acids. Several interesting observations have been made about the two proteins: native conformations are important for the sweet taste, although both proteins are intensely sweet, there are no statistically significant sequence similarities between them; and despite the absence of sequence similarity, antibodies against thaumatin compete for monellin (as well as many other sweet compounds, but not for chemically modified non-sweet monellin) and vice versa. To understand the structural basis of these observations we determined the crystal structure of thaumatin, and report here the structure of monellin at 3 A resolution. Monellin consists of two peptide chains, the A chain of 44 residues and the B chain of 50 residues. We find no similarity between the backbone structure of monellin and that of thaumatin.

Three-dimensional structure of thaumatin I, an intensely sweet protein.

Thaumatin and monellin are the two sweetest compounds known to man--about 100,000 times sweeter than sugar on a molar basis and 3000 times on a weight basis. These proteins represent a unique class of proteins that are taste-active. We report the three-dimensional structure of thaumatin I at 3.1 A resolution.