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Abstract One of the sweetest proteins found in tropical fruits (with a threshold of 50 nM), thaumatin, is also used commercially as a sweetener. Our previous study successfully produced the sweetest thaumatin mutant (D21N), designated hyper-sweet thaumatin, which decreases the sweetness threshold to 31 nM. To investigate why the D21N mutant is sweeter than wild-type thaumatin, we compared the structure of the D21N mutant solved at a subatomic resolution of 0.93 Å with that of wild-type thaumatin determined at 0.90 Å. Although the overall structure of the D21N mutant resembles that of wild-type thaumatin, our subatomic resolution analysis successfully assigned and discriminated the detailed atomic positions of side-chains at position 21. The relative B -factor value of the side-chain at position 21 in the D21N mutant was higher than that of wild-type thaumatin, hinting at a greater flexibility of side-chain at 21 in the hyper-sweet D21N mutant. Furthermore, alternative conformations of Lys19, which is hydrogen-bonded to Asp21 in wild-type, were found only in the D21N mutant. Subatomic resolution analysis revealed that flexible conformations at the sites adjacent to positions 19 and 21 play a crucial role in enhancing sweet potency and may serve to enhance the complementarity of electrostatic potentials for interaction with the sweet taste receptor. Graphical abstract Image 1 Highlights • Subatomic resolution structures of hyper-sweet (D21N) thaumatin and wild-type thaumatin were determined. • Subatomic resolution analysis successfully assigned and discriminated the atomic positions. • Both Lys67 and Arg82 were modelled in two alternative conformations for the first time. • Flexible conformations at the sites adjacent to 19 and 21 might play an important role in enhancing sweetness. [ABSTRACT FROM AUTHOR]

• Structure of thaumatin under acidic conditions (pH 4.0) was determined. • At pH 4.0, the Tm of thaumatin was substantially lower than that at pH 6.0. • Relative B -factor values illustrate the changes in structural features due to a pH change. • The reduction in relative flexibility might play an important role in preventing thermal aggregation. 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. [ABSTRACT FROM AUTHOR]

Thaumatin, an intensely sweet-tasting protein used as a sweetener, elicits a sweet taste at 50 nM. Although two major variants designated thaumatin I and thaumatin II exist in plants, there have been few dedicated thaumatin II structural studies and, to date, data beyond atomic resolution had not been obtained. To identify the detailed structural properties explaining why thaumatin elicits a sweet taste, the structure of recombinant thaumatin II was determined at the resolution of 0.99 Å. Atomic resolution structural analysis with riding hydrogen atoms illustrated the differences in the direction of the side-chains more precisely and the electron density maps of the C-terminal regions were markedly improved. Though it had been suggested that the three consecutive glycine residues (G142-G143-G144) have highly flexible conformations, G143, the central glycine residue was successfully modelled in two conformations for the first time. Furthermore, the side chain r.m.s.d. values for two residues (R67 and R82) critical for sweetness exhibited substantially higher values, suggesting that these residues are highly disordered. These results demonstrated that the flexible conformations in two critical residues favoring their interaction with sweet taste receptors are prominent features of the intensely sweet taste of thaumatin. [ABSTRACT FROM AUTHOR]

Abstract: Thaumatin, a sweet-tasting plant protein, elicits a sweet taste sensation at 50 nM in humans but not rodents. Although it was shown that the cysteine-rich domain (CRD) of human T1R3 (hT1R3) is important for the response to thaumatin, the amino acid residues within CRD critical for response are still unknown. A comparison of the amino acid sequence (69 amino acid residues) of CRD between hT1R3 and mouse T1R3 (mT1R3) revealed sixteen amino acids that differ. In the present study, we converted each of these sixteen amino acids in hT1R3 to their mouse counterpart and examined the response to thaumatin and sucralose using a cell-based assay. No significant decrease in the response to sucralose was seen among any of the sixteen mutants. However, five mutants (Q504K, A537T, R556P, S559P, and R560K) exhibited a significantly diminished response to thaumatin. The five critical residues involved in the response to thaumatin were dispersed in the CRD of hT1R3 and widely distributed when compared to brazzein. The unique intense sweet-taste of thaumatin might be attributed to the different receptor activation mechanism compared to the small molecule sweetener sucralose. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Thaumatin, an intensely sweet-tasting protein, elicits a sweet-taste sensation at a level as low as 50nM. Although previous sensory analyses have suggested that Lys67 and Arg82 are important to the sweetness of thaumatin, the exact effects of each residue on sweet receptors are still unknown. In the present study, various mutants of thaumatin altered at Arg82 as well as Lys67 were prepared and their sweetness levels were quantitatively evaluated by cell-based assays using HEK293 cells expressing human sweet receptors. Mutations at Arg82 had a more deteriorative effect on sweetness than mutations at Lys67. Particularly, a charge inversion at Arg82 (R82E) resulted in an abolishment of the response to sweet receptors even at a concentration as high as 1mM. These results indicate that Arg82 plays a central role in determining the sweetness of thaumatin. A strict spatial charge location at residue 82 appears to be required for interaction with sweet receptors. [Copyright &y& Elsevier]

Abstract: Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27Å. 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. [Copyright &y& Elsevier]

Abstract: Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2–T1R3. Chimeric human– mouse sweet receptors were constructed and their responses to sweeteners were investigated. The human (h) T1R2– mouse (m) T1R3 combination responded to sucralose but not to thaumatin, clearly indicating that a T1R3 subunit from humans is necessary for the interaction with thaumatin. Furthermore, results obtained from using chimeric T1R3s showed that the cysteine-rich domain (CRD) of human T1R3 is important for the interaction with thaumatin. The CRD of T1R3 would be a prominent target for designing new sweeteners. [Copyright &y& Elsevier]

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness. [ABSTRACT FROM AUTHOR]

Abstract: Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the α-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis. [Copyright &y& Elsevier]

Most proteins are tasteless and flavorless, while some proteins elicit a sweet-taste response on the human palate. Six proteins, thaumatin, monellin, mabinlin, brazzein, egg lysozyme, and neoculin (previously considered as curculin) have been identified as sweet-tasting proteins. However, no common features among them have been observed. Herein, recent advances in the research of sweet-tasting proteins and the production of such proteins by biotechnological approaches are reviewed. Information on the structure-sweetness relationship for these proteins would help not only in the clarification of the mechanism of interaction of sweet-tasting proteins with their receptors, but also in the design of more effective low-calorie sweeteners. [Copyright &y& Elsevier]