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Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains: an A chain of 44 amino acid residues and a B chain of 50 residues. Microbial transglutaminase (MTGase) was used for ligation of the monellin subunits without any protecting groups, and without activation of the C alpha-carboxyl group at the C-terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the gamma-amide group of the Gln residue and the epsilon-amino group of Lys, a monellin B chain analogue in which LLQG was elongated at the C-terminus (B-LLQG) was synthesized by solid-phase synthesis. The monellin A chain analogue in which KGK was elongated at the N-terminus (KGK-A) was synthesized by the same method as that of the B chain analogue. The KGK-A chain and the B-LLQG chain were coupled by MTGase to give single-chain analogue of monellin. The single-chain analogue of monellin was characterized by analytical reverse phase high performance liquid chromatography, electrospray ionization, and amino acid analyses. All analyses gave satisfactory results. The single-chain analogue of monellin was more heat stable than natural monellin.

The racemic mixture of synthetic d and l-monellin has been crystallized, and its structure has been determined by X-ray crystallography at 1.9 A resolution. The crystal structure consists of two d and two l-monellin molecules in the P1 unit cell with a pseudo-centrosymmetrical arrangement. The final structure reveals small but significant structural differences between d and l-monellin in the same crystal. Possible reasons for these differences and their implications are discussed., (Copyright 1999 Academic Press.)

The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste.

Single-chain monellin, a sweet protein, was synthesized by stepwise solid-phase synthesis. After deprotection, and purification with HPLC, single-chain monellin was obtained in a yield of 0.3% based on the starting resin.

The sweetness-suppressing polypeptide gurmarin isolated from Gymnema sylvestre consists of 35 amino acid residues and includes three intramolecular disulfide bonds. The roles of the three disulfide bonds were investigated by replacing each with two alanine residues by solid-phase synthesis. Nine analogues of [Ala3,18]gurmarin, [Ala10,23]gurmarin, and [Ala17,33]-gurmarin were obtained. Three analogues had native disulfide bonds, while the other six had non-native disulfide bonds. The three analogues with native disulfide bonds suppressed the response to sucrose, but not those to glucose, fructose, saccharin, or glycine in rats. In contrast, the six analogues with non-native disulfide bonds did not suppress the responses to any of these sweeteners. These results suggest that the native disulfide bonds of gurmarin are necessary for interaction with the receptor protein, and that the sucrose-specific receptor site is present in rats.

The D-enantiomer of a potently sweet protein, monellin, has been crystallized and analyzed by X-ray crystallography at 1.8 A resolut ion. Two crystal forms (I and II) appeared under crystallization conditions similar, but not identical, to the crystallization conditions of natural L-monellin. There are four molecules per asymmetric unit in crystal form I and one in crystal form II. Crystal form I is not reproducible and is equivalent to that of monoclinic L-monellin. Intermonomer contacts in crystal form II are very different from those found in natural L-monellin crystals. The backbone trace of D-monellin resembles very closely the mirror image of that of L-monellin, but the N- and C-terminus backbones as well as several side-chain conformations of D-monellin are different from those of natural L-monellin. Most of these apparent differences may be attributable to the crystal packing differences.

The sweetness-suppressing polypeptide gurmarin isolated from Gymnema sylvestre consists of 35 amino acid residues and contains three intramolecular disulfide bonds. Nuclear magnetic resonance analysis showed that the hydrophobic side chains of Tyr-13, Tyr-14, Trp-28, and Trp-29 in gurmarin are oriented outwardly. Together with the hydrophobic side chains of Leu-9, Ile-11, and Pro-12, they form a hydrophobic cluster, and therefore these hydrophobic groups are assumed to act as the site for interaction with the receptor protein. To examine the roles of these hydrophobic amino acids, they were replaced by Gly. The resulting [Gly13,14,28,29] gurmarin and [Gly9,11,13,14,28,29]-gurmarin did not suppress the responses to sucrose, glucose, fructose, or Gly. This result strongly suggests that these hydrophobic amino acids are involved in the interaction with the receptor protein.

Monellin, an intensely sweet protein and a non-sweet analog in which the AspB7 in monellin has been replaced with AbuB7 were studied by NMR. The results of our investigations show that the 3-dimensional structure of these two proteins are very similar indicating that the lack of the beta-carboxyl group in the AbuB7 analog is responsible for the loss of sweet potency. Selectively labeled monellin was prepared by solid-phase peptide synthesis by incorporating 15N-labeled amino acids into 10 key positions including AspB7. The internal mobility of these 10 key residues in monellin was estimated by the method of model-free analyses and our NMR studies show that AspB7 is the most flexible of these 10 residues. The flexibility of the AspB7 side chain may be important for receptor binding.

The sweetness-suppressing polypeptide gurmarin isolated from the leaves of Gymnema sylvestre consists of 35 amino acid residues including three intramolecular disulfide bonds. Herein, the total chemical synthesis of gurmarin was performed by the stepwise fluoren-9-ylmethoxy-carbonyl solid-phase method, the yield of reduced gurmarin being 1.9% based on the starting amino acid resin. Disulfide formation was carried out in the presence of a redox system of reduced glutathione/oxidized glutathione to give gurmarin in a yield of 35.5%. The product was identical to natural gurmarin by analytical reverse phase high performance liquid chromatography (RP-HPLC), mass spectroscopy (MS), and peptide mapping, and suppressed the responses to sucrose, D-glucose, and L-glucose in a rat. The D enantiomer (all D-amino acid gurmarin) was also synthesized, and was shown to be the mirror image of gurmarin. Interestingly, the D enantiomer (ent-gurmarin) also suppressed the responses to sucrose, D-glucose, and L-glucose in a rat.

The sweet protein brazzein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon is 2000-500 times sweeter than sucrose and consists of 54 amino acid residues with four intramolecular disulfide bonds. Brazzein was prepared by the fluoren-9-yl-methoxycarbonyl solid-phase method, and was identical to natural brazzein by high performance liquid chromatography, mass spectroscopy, peptide mapping, and taste evaluation. The D enantiomer of brazzein was also synthesized, and was shown to be the mirror image of brazzein. The D enantiomer (ent-brazzein) was devoid of any sweetness and was essentially tasteless.

The thermostable sweet protein brazzein consists of 54 amino acid residues and has four intramolecular disulfide bonds, the location of which is unknown. We found that brazzein resists enzymatic hydrolysis at enzyme/substrate ratios (w/w) of 1:100-1:10 at 35-40 degrees C for 24-48 h. Brazzein was hydrolyzed using thermolysin at an enzyme/substrate ratio of 1:1 (w/w) in water, pH 5.5, for 6 h and at 50 degrees C. The disulfide bonds were determined, by a combination of mass spectrometric analysis and amino acid sequencing of cystine-containing peptides, to be between Cys4-Cys52, Cys16-Cys37, Cys22-Cys47, and Cys26-Cys49. These disulfide bonds contribute to its thermostability.

The sweetness-suppressing polypeptide gurmarin has been isolated from the leaves of Gymnema sylvestre and consists of 35 amino acid residues including three intramolecular disulfide bonds. The primary structure has already been determined. The positions of the disulfide bonds were located, by a combination of mass spectrometric analysis and sequencing of cystine-containing peptides obtained by thermolysin-catalyzed hydrolysis of gurmarin, to be at Cys3-Cys18, Cys10-Cys23, and Cys17-Cys33.

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Synthetic monellin is 4000 times as sweet as sucrose on a weight basis, and the native conformation is essential for the sweet taste. Knowledge of the active site of monellin will provide important information on the mode of interaction between sweeteners and their receptors. If the replacement of a certain amino acid residue in monellin removes the sweet taste, while the native conformation is retained, it may be concluded that the position replaced is the active site. Our previous replacement studies on Asp residues in the A chain did not remove the sweet taste. The B chain contains two Asp residues at positions 7 and 21, which were replaced by Asn. [AsnB21]Monellin and [AsnB7]monellin were 7000 and 20 times sweeter than sucrose, respectively. The low potency of the [AsnB7]monellin indicates that AspB7 participates in binding with the receptor. AspB7 was then replaced by Abu. [AbuB7]Monellin was devoid of sweetness, and retained the native conformation. AspB7 is located at the surface of the molecule (Ogata et al.). These results suggest that Asp7 in the B chain is the highly probable active site of monellin.

In an attempt to delineate the binding site(s) of monellin to the receptor by means of a structure-taste relationship, we synthesized four monellin analogues, [AsnA16]-, [AsnA22]-, [GlnA25]-, and [AsnA26]-monellin, which were 7500, 750, 2500, and 5500 times as sweet as sucrose on a weight basis, respectively. Among them, [AsnA22]monellin and [GlnA25]monellin were less sweet than monellin, and were susceptible to the HPLC conditions used. It can be concluded that Asp16, Asp22, Glu25, and Asp26 residues of the A chain did not participate in binding with the receptor, since the sweet taste was not removed by replacing the amino acid residues with Asn or Gln. It can also be concluded that Asp22 and Glu25 of the A chain may have participated in intramolecular binding, as was pointed out by Kim et al., since exchanging Asp22 and Glu25 of the A chain with Asn and Gln significantly decreased the stability in solution.

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Since blocking the free SH group of Cys41 in the B chain or treating the adjacent Met42 with CNBr removed its sweetness, this part of the molecule has been suggested to be essential for the sweetness. The [Ser41] B chain, an analogue of the B chain, was synthesized by the stepwise Fmoc solid-phase method in an overall yield of 1.9%. The synthetic B chain analogue was combined with the A chain, which was left over from a previous work, to give [Ser41] B-chain monellin in a yield of 31.0%. This synthetic monellin analogue was 2000 times as sweet as sucrose. Changing the Cys41 residue to the Ser residue significantly decreased the sweetness potency and stability of the molecule in solution. Crystallization was carried out by a vapor diffusion method.

The sweet protein, monellin, consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. The B chain was synthesized by the stepwise Fmoc solid-phase method in an overall yield of 6.2%. The synthetic B chain was combined with the synthetic A chain, which was left over from a previous work, to give monellin in a yield of 25.7%. The synthetic monellin was approximately 4000 times as sweet as sucrose, while the previously synthesized [Asn49, Glu50] B-chain monellin was also approximately 4000 times as sweet as sucrose. Exchanging Glu49 and Asn50 in the B chain did not affect the sweetness potency. Crystallization was performed by a vapor diffusion method.

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. The [Asn22, Gln25, Asn26]-A chain, an anologue of the A chain, was synthesized by the stepwise Fmoc-solid-phase method in an overall yield of 13.4%. The synthetic A chain analogue was combined with the [Asn49, Glu50]-B chain, which was left over from a previous work, to give [Asn22, Gln25, Asn26]-A-chain-[Asn49,Glu50]-B-chain-monellin in a yield of 26.2%. This synthetic monellin analogue was approximately 550 times sweeter than sucrose. Changing the carboxyl groups of Asp22, Glu25, and Asp26 of the A chain to amide groups significantly decreased the sweetness potency. Crystallization was performed by a vapor diffusion method.

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of these chains. The complete amino acid sequence of monellin was determined by a combination of FAB- and ESI-mass spectrometry, and by automatic Edman degradation.

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of the A and B chains. The A and B chains corresponding to one of the reported monellin structures were synthesized by the stepwise solid-phase method using the Fmoc strategy in overall yields of 14.1% and 5.6%, respectively. The characterization of the synthetic peptides by HPLC, FAB-MS, amino acid analysis and sequencing fully supported the expected structures. The individual synthetic A and B chains were not sweet. Combination of the two chains, and subsequent HPLC purification gave monellin in a yield of 53.9%. The synthetic monellin had a distinct, lingering sweet taste (4000 times sweeter than sucrose) and was crystallized by a vapor diffusion method. The synthetic product was identical to natural monellin by HPLC, but not by tryptic mapping. These results indicate that the reported structure for monellin differs slightly from that of natural monellin.