A Novel Structural Type of Sweet Proteins and the Main Structural Basis for Its Sweetness

Available online at www.sciencedirect.com Journal of Structural Biology Journal of Structural Biology 162 (2008) 50–62 www.elsevier.com/locate/yjsbi

Crystal structure of Mabinlin II: A novel structural type of sweet proteins and the main structural basis for its sweetness

De-Feng Li a, Peihua Jiang b, De-Yu Zhu a, Yonglin Hu a, Marianna Max b, Da-Cheng Wang a,*

a National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, People’s Republic of China b Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029, USA

Received 22 July 2007; received in revised form 23 October 2007; accepted 12 December 2007 Available online 11 January 2008

Abstract

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7 A˚ resolution by the SIRAS method. The Mab II 3D structure features in an ‘‘all alpha” fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54–B64 was found in Face B, which adopts a special sequence, NL–P–NI–C–NI–P–NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model. Ó 2008 Elsevier Inc. All rights reserved.

Keywords: Sweet protein; Mabinlin; Crystal structure; Sweet receptor; hT1R2/T1R3-Mab II interaction; Structural basis of sweet taste

1. Introduction increase by 1.5–2 million per year (Zhang, 2004). A series of artificial sweeteners like Saccharin, Aspartame, and Sweet proteins are low-calorie sweetener (Kant, 2005). Cyclamate are used world wide as low calorie replacements In recent years, the prevalence of the diseases related to for these patients. So far the most popular sweeteners are the consumption of sugar, such as obesity, diabetes, hyper- small molecule compounds, which may incur some site glycemia, and caries, has increased dramatically. For exam- effects such as psychological problems, mental disorders, ple, the number of diabetes mellitus patients in China has bladder cancer, and heart failure (Weihrauch et al., 2002; been estimated at 50 million in 2004 and it is forecast to Kanarek, 1994; Cohe, 2001; Nabors, 1988; Hagiwara et al., 1984). Instead, sweet proteins can be used as natural, low calorie sweeteners without triggering a demand for * Corresponding author. Fax: +86 10 64888560. insulin in these patients whereas sucrose does. Therefore, E-mail address: [email protected] (D.-C. Wang). they are potential replacements for artificial sweeteners

1047-8477/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2007.12.007 D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62 51 and sugars and may find applications in food and beverage from these seeds (Hu and He, 1983; Hu et al., 1985a,b). industries. Among others, Mab II possesses the most interesting prop- Until now, five sweet proteins have been identified, erties. Its sweetness is estimated to be around 100 times namely thaumatin (Van der Weld and Loeve, 1972), monel- that of sucrose on weight basis (Hu and He, 1983) and lin (Morris and Cagan, 1972), brazzein (Ding and Hellek- remains unchanged after 48-h incubation at boiling tem- ant, 1994), neoculin (Shirasuka et al., 2004; Suzuki et al., perature (Ding and Hu, 1986). It is now known that Mab 2004) (formerly known as curculin (Yamashita et al., II consists of an A-chain with 33 amino acid residues and 1990)), and mabinlin (Hu and He, 1983). Among them, a B-chain with 72 amino acid residues and has a total the 3D structures of thaumatin (Ogata et al., 1992), monel- molecular weight of 12.4 KDa (Liu et al., 1993). The B- lin (Somoza et al., 1993), neoculin (Shimizu-Ibuka et al., chain contains two intrachain disulfide bonds and is cross- 2006), and brazzein (Caldwell et al., 1998) have been deter- linked with the A-chain through two interchain disulfide mined. Besides, by using the site-directed mutagenesis anal- bridges (Nirasawa et al., 1994). The full sequence of Mab ysis a series of residues related to the sweet activity have II shows no obvious homology with that of the other sweet been identified for specific protein sweeteners, including proteins reported so far. Therefore, it is interesting to elu- monellin (Mizukoshi et al., 1997; Somoza et al., 1995), cidate the 3D structure of Mab II and identify the unique thaumatin (Kaneko and Kitabatake, 2001), and brazzein structural elements for eliciting the sweetness for in-depth (Assadi-Porter et al., 2000; Jin et al., 2003a,b). investigation of its structure–function relationship, which On the other hand, the human receptors for sweet taste, may benefit the development of a useful sweetener based hT1R2/T1R3, have been identified recently (Li et al., on Mab II. 2002). Meanwhile Temussi proposed a ‘‘wedge model” (Temussi, 2002) for the interactions between sweeteners 2. Experimental procedures and T1R2–T1R3 receptor. More recently, Temussi and coworkers built all four possible forms of the extracellular 2.1. Purification heterodimeric sweet receptor by homology modeling to perform docking with sweet proteins, including brazzein, The Mab II protein used in crystallization was purified monellin, and thaumatin and described full account of from the mature seeds of C. masaikai plants grown in the the ‘‘wedge model” for sweet proteins (Morini et al., south of Yunnan Province, China, according to the method 2005). Some recent reports on sweet proteins, such as neo- described previously (Hu and He, 1983). culin (Shimizu-Ibuka et al., 2006), brazzein (Walters and Hellekant, 2006) also demonstrated the validity of the ‘‘wedge model”. However, there are no significant similar- 2.2. Crystallization ities in either amino acid sequences or three-dimensional structures in all sweet proteins reported. Moreover, no All crystallization experiments were performed with the common structural basis for sweetness has been identified hanging-drop vapor-diffusion method. Mab II was dis- from these proteins. Therefore it is significant to find new solved in pure water at a concentration of 10 mg/ml for type of sweet protein for understanding their structural crystallization. The crystallization was achieved in a rather diversities. Here we report the X-ray crystal structure of unique condition of high temperature at 310 K. The drops a highly thermo-stable sweet protein Mabinlin II (Mab were formed by mixing 2 ll of protein solution with 2 llof II), which shows a novel, all alpha fold with two polypep- reservoir solution and equilibrated against 400 ll reservoir tide chains crosslinked by four disulfide bridges. A docking solution in each well. The best crystals were obtained with study of Mab II onto the model of sweet receptor hT1R2/ a reservoir solution (400 ll) containing 0.1 M sodium ace- hT1R3 revealed the main structural basis for the Mab II- tate trihydrate buffer (pH 4.6) and 1.4 M sodium malonate hT1R2/T1R3 interaction to elicit its sweet taste. Besides, (Li et al., 2006). an interesting functional ramification of A- and B-chains in eliciting the sweet taste of Mab II was also observed. 2.3. Data collection and processing The results provided another example of the validity of the ‘‘wedge model”. All diffraction data were collected on a Rigaku R-Axis Mabinlin II is isolated from the seeds of mabinlang, the IV++ image plate using CuKa radiation (k = 1.5418 A˚ ) plant of Capparis masaikai Levl. growing in the subtropical from a rotating anode operating at 40 kV and 20 mA with region of the Yunnan province of China and bearing fruits 0.1 mm confocus incident beam diameter. The crystals of tennis-ball size (Hu and He, 1983). The mature seed of were briefly soaked in paraffin oil (Hampton Research) mabinlang elicits a sweet taste with a long aftertaste. The after being mounted in nylon cryoloops (Hampton natives chew the seeds for a unique sweet perception with Research) and then flash-cooled in a nitrogen-gas stream a long aftertaste. Immediately after chewing, a bitter and at 85 K. The native data were collected with a crystal-to- astringent stimulus is left, followed by a prolonged sensa- detector distance of 100 mm, Du =1° and 300 s exposure tion of sweetness. Hu and his coworkers isolated four sweet time. A total of 180 frames were collected. MOSFLM proteins, named Mabinlin I, II, III, and IV, respectively, (V6.2.3), TRUNCATE, and SCALA from CCP4 program 52 D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62 suite V.4.2.2 (CCP4, 1994) were used for processing, reduc- Table 1 ing, and scaling of the diffraction data. Data collection, phasing, and refinement statistics of Mab II Native KI 2.4. Structure determination and refinement A. Data collection Space group (molecules/asu) C2 C2 The structure of Mab II was determined by the SIRAS a (A˚ ) 80.11 80.38 ˚ method using iodine derivative prepared through the b (A) 51.08 51.44 c (A˚ ) 47.34 48.22 cryo-soaking approach (Dauter et al., 2000). To obtain b (°) 122.77 122.56 suitable heavy atom derivatives, the native crystals were Wavelength (A˚ ) 1.5418 1.5418 soaked for 24 h in the mother liquor containing KI reagent Resolution range (A˚ ) 20.35–1.70 25.74–2.2 at 0.1 M concentration. The heavy atom derivative data Observation (I/r(I) > 0) 62800 60934 were collected with a crystal-to-detector distance of Unique reflections (I/r(I) > 0) 17686 8469 Redundancy 3.6 7.2 150 mm, Du =1° and 300 s exposure time. A total of 360 Last shell (A˚ ) 1.79–1.70 2.32–2.20 frames were collected. Eighteen possible iodine sites were Rsym (%): overall (last shell) 5.3(26.6) 6.4(16.6) found by ShelxD (Schneider and Sheldrick, 2002) using hI/r(I)i: overall (last shell) 6.0(2.6) 6.6(2.2) the SIRAS method. The program SHARP (De La Fortelle Completeness (%): overall (last shell) 99.5(100) 99.5(97.1) and Bricogne, 1997) was used for phase calculation and Solvent content 25% 25% density modification. The model was manually built using B. Phasing the program O (Jones et al., 1991). The structure was Iodine atom sites 16 Resolution range of data used (A˚ ) 20.35–3.0 refined to a resolution of 1.7 A˚ with R-factor of 0.221 Rcullis Iso Ano and Rfree of 0.254 using CNS version 1.1 (Brunger et al., 0.806 0.663 1998). There are two Mab II molecules in an asymmetric Phasing power Iso Ano unit. The final model contains 189 ordered residues (A4– 1.093 1.610 A28, B4–B69, C2–C29, and D4–D71), one acetate molecule Density modification, FOM 0.69 and 125 water molecules in the asymmetric unit. The ste- C. Structure refinement reochemical assessments of the structure were performed Resolution range (A˚ ) 20.35–1.70 by PROCHECK (Laskowski, 2001). The statistics data Sigma cutoff 2.0 R-factor (%) 22.1 are listed in Table 1. Rfree (%) 25.4 Ramachandran statistics (%) 2.5. 3D-docking with sweet receptor Most favoured regions 95.7 Additionally allowed regions 4.3 To explore the possible interaction between Mab II and Generously allowed regions 0 Disallowed regions 0 hT1R2/T1R3, a 3D-docking computation was performed. Numbers of protein non-H atoms 1547 Prior to calculation of the docking model, a structural Numbers of solvent molecules 125 H2O+1Ac model of the sweet-taste receptor hT1R2/T1R3 was built Average B value (A˚ 2)37 by homology modeling based on the crystal structure of the extracellular region of the metabotropic glutamate two models for the complexed active closed–open form, receptor 1 (mGluR), also called ligand-binding region of henceforth dubbed Aoc_AB (T1R2 modeled on chain A mGluR1 (m1-LBR) (Kunishima et al., 2000). At present, and T1R3 modeled on chain B of 1ewk) and Aoc_BA the crystal structures of m1-LBR in two unliganded forms, (T1R3 modeled on chain A and T1R2 modeled on chain called free forms I (PDB entry: 1EWT) and II (PDB entry: B of 1ewk). 1EWV), and in a complex form with glutamate (PDB First, the Aoc_AB model was modeled. The free form II entry: 1EWK) were determined (Kunishima et al., 2000). structure of extracellular domain of mGluR (m1-LBR, Free form I and II are the inactive or resting open–open PDB entry: 1EWV, not 1EWK) was used as a template. form of mGluR and the active closed–open form of The primary sequences of T1R2 and T1R3 were obtained mGluR, respectively. The structure of free form II is almost from the Swiss-Prot database (entry codes: TS1R2_HU- identical to that in complex with glutamate and so is con- MAN and TS1R3_HUMAN). Sequences of T1R2, sidered to be similar with the active conformation of sweet T1R3, and m1-LRB were aligned by CLUSTAL (Thomp- protein receptor (Temussi, 2002). Considered that T1R2/ son et al., 1994). The two chains of the initial ml-LBR T1R3 is a heterodimer while mGluR is a homodimer, as molecular model were then replaced by T1R2 and T1R3, Morini et al. described there are total of four T1R2/ respectively, and the resultant complex was refined by T1R3 models should be modeled, (Morini et al., 2005), Modeller 8V2 (Kelley et al., 2000). The other three models including two models for the inactive open–open form, of T1R2/T1R3 (Roo_AB, Roo_BA, Aoc_BA) were built henceforth dubbed Roo_AB (where AB means T1R2 mod- up with the same procedure. The four models were then eled on chain A and T1R3 modeled on chain B of the 1ewt used in 3D-docking of Mab II molecule. template) and Roo_BA (T1R3 modeled on chain A and The docking of Mab II with the hT1R2/T1R3 complex T1R2 modeled on chain B of the 1ewt template), and was performed using the program GRAMM v.1.0.3 (Vak- D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62 53 ser, 1995; Vakser et al., 1999; Tovchigrechko and Vakser, cium imaging experiments with the expressed hT1R2/ 2005). The representative structure at each protonation hT1R3 cell model (Jiang et al., 2004, 2005). For the exper- state was docked into the hT1R2/T1R3 receptor model. iments, cDNAs encoding hT1R2, hT1R3, and Ga16gust44 First, docking was evaluated only through energy scores. (Jiang et al., 2004, 2005) were transiently transfected into Then, the models taking the highest score with the lowest HEK293E cells using lipofectamine 2000 (Invitrogen) energy of Mab II-hT1R2/T1R3 were built and evaluated according to the manufacturer’s protocol. After 4 h, cells manually. were trypsinized and seeded onto poly-lysine coated 96- well plates at 40000 cells/well and cultured in low-glucose 2.6. Separation of two chains of Mab II DMEM supplemented with GlutaMAX-1 and 10% dia- lyzed FBS (Invitrogen). After an additional 40 h, cells were To verify the main structural basis of Mab II for the loaded with the calcium dye Fluo-4 (Invitrogen), dissolved sweet taste, which was proposed from the docking test, in Dulbecco’s PBS buffer (DPBS, Invitrogen) at a concen- and distinguish the functional role of A-chain from that tration of 3 lM, for 1 h at room temperature. After three of B-chain, the two polypeptide chains of Mab II were sep- washes with DPBS, stimulation was performed with DPBS arated. Mab II protein powder was dissolved in 6 M guani- supplemented with tastants. Calcium mobilization was dine hydrochloride with 5 mM b-mercaptoethanol at a recorded using an Olympus Fluoview confocal microscope. concentration of 5 mg/ml and incubated for 4 h at 310 K. The HEK293E cells coexpressed hT1R2/T1R3 were stimu- Two peaks were collected after elution from a C18- lated with the purified Mab II (0.5%), Mab-A (0.05%), reverse-phase column (5 lm, 4.6 250 mm) on an AKTA Mab-B (0.5%), and D-tryptophan (10 mM) as a control. Purifier system (Amersham Pharmacia Biotech) and then Data acquisition and analysis of time series images were lyophilized. These two peaks were verified as the A- and performed using the software Fluoview version 2.1. Every B-chains of Mab II by MALDI-TOF mass spectra. experiment was repeated for at least three times.

2.7. Fluorescence emission spectra of Mab II and Mab-B 2.10. Sweet activity test To compare the conformational state of the separated The sweetness of Mab II, Mab-A, and Mab-B, were B-chain (Mab-B) with that in the native Mab II, Mab-B, evaluated by a regular procedure (Hu and He, 1983). Five and Mab II were subjected to fluorescence emission spec- milligrams per milliliter solutions of Mab II, Mab-A, Mab- trometric analysis. Because there is only one tryptophan B, and a blank solution (pure water) were prepared. The residue located at B-chain (TrpB72) in Mab II, the fluores- samples were blindly taken into the test. Each time 10 ll cence emission spectra of this residue can be used to mon- of the solution was dropped on the tongues of the testers itor the folding status of the B-chain in comparison with (Hu and He, 1983) have been trained by 0.3 M sucrose that of the native Mab II (Hu et al., 1985a,b). For the solution. After about 10 s, the testers identified whether experiment, 1 mg/ml Mab II and Mab-B solutions were the solutions were sweet and the results were recorded to prepared. Their intrinsic fluoresce were measured with exci- identify which solutions were sweet. Total three people tation at 280 nm and slits of 5 nm. The results were were chosen as the tester and every experiment was recorded at room temperature using a Shimadzu repeated for at least three times. RF5301PC spectrofluorometer.

2.8. 1D 1H NMR spectrum 3. Result and discussion

The 1D 1H NMR experiment was taken further to check 3.1. Overall structure of Mab II in crystals the fold state of the separated B-chain. The NMR sample used in the experiment contains 0.5 mM Mab-B protein, The final Mab II model contains two Mab II molecules and 10% (v/v) D2O in 50 mM phosphate buffer, pH 7.0. in an asymmetric unit (Fig. 1), which are related by a non- 1D 1H NMR experiments were performed at 310 K on a crystallographic (NCS) twofold rotation axis nearly paral- Bruker DMX 600 MHz spectrometer equipped with a lel with the b axis of the unit cell (Fig. 1a). The general cryo-probe. folds of the two Mab II molecules are similar to each other with an rmsd of 0.464 A˚ for main chain atoms. These two 2.9. Test of interactions between Mab molecules and human NCS-related molecules contact each other by their respec- sweet receptor tive residues B34–B40 in an antiparallel arrangement through a well-ordered hydrogen-bond network (Fig. 1b). Heterologously expressed hT1R2 + hT1R3 has been Their surfaces in this contact region display a high degree shown to respond to many sweeteners, including monellin, of shape complementary, exhibiting a shape coefficient thaumatin, brazzein, and other sweet compounds (Jiang (Lawrence and Colman, 1993) of 0.757 (Sc = 1.0 for inter- et al., 2004). The response of the human sweet receptor face with geometrically perfect fits), and a buried surface to Mab II, Mab-A, and Mab-B was tested through the cal- area of 435 A˚ 2 as calculated by SURFACE (Chothia, 54 D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62

Fig. 1. Overall structure of Mab II in an asymmetric unit. (a) Ribbon presentation of two Mab II molecules in the asymmetric unit viewing from the local twofold axis. (b) The stereo drawing of interface between the two molecules showing a well-ordered hydrogen-bond network formed by B34–B40, respectively, in the two molecules with an antiparallel arrangement. W stands a water molecule. The ﬁgures were prepared by MolScript.

1975) (the total surface area of a Mab II molecule is so far. The double chain structure of Mab II belongs to ‘‘all 5506 A˚ 2). alpha protein” in SCOP classiﬁcation (Murzin et al., 1995), which is distinct from all known structures of the sweet 3.2. A novel structure type of sweet protein proteins featuring beta or alpha/beta type. Thus, the 3D structure of Mab II presents a novel structure type of the The full sequence of the monomeric Mab II molecule sweet proteins (Table 2). The molecule Mab II is the only contains 105 residues (Fig. 2a), which shows no signiﬁcant sweet protein that consists of two covalent-linked polypep- homology with that of all the other sweet proteins reported tide chains. These two chains are crosslinked by two inter- D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62 55

Fig. 2. Unique structure of Mab II molecule. (a) Sequence and secondary structure distribution of Mab II. (b) The stereo drawing of the general fold of Mab II molecule. The unique segment B54–B64 is highlighted by a circle. (c) Topology diagram of the unique [NL/I] tetralet motif. (d) Space-ﬁlling model of Mab II. The side chains of Asn residues from B54, 57, 60, and 63 of the unique motif are highlighted, which are rooted in a hydrophobic cluster and presented on the ligand-accessible surface. (e) Structure of the [NL/I] tetralet motif in the orientation same as (d). The side chains only involved in the motif are shown. All side chains of hydrophobic residues are shown in green stick. All O and N atoms in drawings are colored in red and blue, respectively. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this paper.) 56 D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62

Table 2 tural unity. In fact, the side chains of AsnB54, AsnB57, Summary of sweet proteins’ classification in SCOP (Murzin et al., 1995) AsnB60, and AsnB63 are all protruded from the specific and CATH (Orengo et al., 1997) surface and ligand-accessible. It is plausible to infer that SCOP CATH this unique structural motif may possess certain critical Mab II All alpha proteins Mainly alpha structure–function roles, which was generally identified Brazzein Small proteins Alpha beta by 3D-docking and experiments as shown below. Monellin Alpha and beta proteins (a + b) Alpha beta Neoculin All beta proteins Mainly beta Thaumatin All beta proteins Mainly beta 3.4. An amphiphilic surface

Mab II molecule has five negatively (three Asp and two chain disulfide bridges (CysA5-CysB21 and CysA18- Glu) and 18 positively (all Arg) charged residues. Among CysB10). In addition, the B-chain has two intrachain disul- them the negative charged residues are scattered on the fide bridges (CysB11-CysB59 and CysB23-CysB67) (Fig. 2a molecular surface, including two Glu (A1/C1 and B1/D1) and b). The A-chain consists of two a-helices (h1, A4–A12; and one Asp (A33/C33) which are disorder and located h2, A15–28) connected with a hydrogen-bonded turn (t1, at the N- and C-terminus. Most of the positively charged A13–A14) to form a helix–turn–helix motif (Fig. 2a and residues are distributed on one side of the exterior surface b). The B-chain has three a-helices (h3, B2–B15; h4, B21– to form a strongly cationic face, Face A (Fig. 3a). While, B37; h5, B41–B59) (Fig. 2a and b). Helices h3 and h4 are most of polar residues (Asn, Gln) with certain hydrophobic connected by a short 310 helix (g1, B16–B20) to form a residues are gathered onto another side of the molecular helix–turn–helix motif. Helices h4 and h5 are connected surface to create a neutral face, Face B (Fig. 3b). It is noted with a short loop (l1, B38–B40). The C-terminus of B-chain that the four characteristic Asn presented by the unique is a coil (c1, B65–B71) and connected to h4 through a b- [NL/NI] tetralet motif described above are just protruded turn (t2, B59–B64). Helix h3, which contains the Cys-Cys from the neutral Face B (Fig. 3b). This unique ‘‘amphi- motif, is linked to h2 and to the C-terminal end of h5 by pathic design” of the molecular surface may be related to disulfide bridges. The N-terminus of h4, which contains the primary interaction of Mab II molecule with its Cys-X-Cys motif, connects to h1 and to the C-terminus receptor. of the protein (c1) (Fig. 2a and b). The overall structure of Mab II molecule is rather compact: the volume of the 3.5. Mab II-hT1R2/T1R3 complex model from 3D-docking protein as calculated with the program VADRA (Wishart suggests that the Mab-B-chain is essential for the Mab et al., 2003) is 12900 A˚ 3, 12% lower than expected from II-receptor interaction its molecular weight (about 14500 A˚ 3). This compactness, together with the existence of four disulfide bridges, may Following the account of the ‘‘wedge model” for sweet contribute to its high thermostability. proteins by Temussi and coworkers (Temussi, 2002; Morini et al., 2005), the 3D-dockings of Mab II interacting with all four possible forms of the extracellular heterodimeric sweet 3.3. A unique structural motif receptor hT1R2/T1R3 were performed. The refined Mab II structure was first docked into the hT1R2/T1R3 Aoc_AB Detailed inspection of sequence found that the segment model. A global exhaustive six-dimensional grid search B54–B64 takes a unique residue arrangement, NLPNIC- through the relative translations and rotations of the mol- NIPNI, which features four successive (Asn-Leu/Ile) dipo- ecules was performed. The energy scores of all possible lar units connected by three conformation-constrained complex models were sorted, and 10 candidates with the residues, two prolines and a cystein involved in disulfide highest score and the lowest energy were built up for the bridge, in between (Fig. 2c). We call this special local struc- further evaluation. Each generated model was then taken ture as [NL or NI] tetralet motif. On the tertiary structure into a manual inspection on its reasonableness from the this motif is located in the C-terminal part of h5 with the b- structural aspect. The detailed stereochemical analysis turn t2 (Fig. 2a and b). The motif adopts a subtle structural showed that, among 10 candidates, five models displayed organization as shown in Fig. 2d and e. Four hydrophobic serious close contacts between Mab II and the receptor in residues of the motif, respectively, take part in the hydro- some areas (Table 3) showing the spatial hindrance, which phobic core of the molecule. As a consequence the four therefore were discarded. The remaining five candidates polar residues Asn are rooted in the hydrophobic clusters were sorted according to their folding energy and contact to be well stabilized. Interestingly, the residues connected areas (Table 3) and the best one in the list, Candidate 1, these Asn-based dipolar units are proline (ProB56, B62) was chosen as the representative model of Mab II- or Cys (CysB59) involved in the disulfide bridge (CysB11- hT1R2/T1R3 complex. CysB59). It is well known that they all are conformation- In this complex model, the Mab II molecule contacts constrained residues. In this way the four Asn residues only with protomer T1R3 of the receptor and fits to should be directed into a specific orientation, respectively, T1R3 structure well with a good stereochemical compli- and structurally related each other to form a tertiary struc- mentary as shown in Fig. 4. There is an area as large as D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62 57

Fig. 3. The amphiphilic surface of Mab II molecule. Most of 18 Arg residues are distributed on the one side of the exterior surface to form a cationic face (Face A) and some neutral residues including Asn, Gln, Leu, and Ile are gathered onto the opposite surface to create a neutral face (Face B). The four Asn residues of the [NL/I] tetralet motif are located on Face B. The ﬁgures were prepared by PyMol.

Table 3 The docking tests of Mab II with the other three models Energy and geometry states of 10 complex models from 3D-docking of T1R2/T1R3, Roo_AB, Roo_BA, and Aoc_BA were Candidate Fold energy Contact area Close contacts also performed to search for the possible interactions in ˚ 2 (kJ/mol) (A ) region between. In the docking of Mab II to the inactive forms 1 929.79 1532 of T1R2/T1R3 (Roo_AB and Roo_BA), a series of dock- 2 928.03 1473 ing models were built, but they all did not show the good 3 925.28 1278 4 923.88 936 charge complementary and the distinct difference in energy, 5 919.73 1357 contact area of surface and molecular orientation. We con- 6 930.46 1635 T1R3 119–137 sidered that a specific binding of Mab to this form of the 7 928.47 1379 T1R3 1–25 receptor seems unlikely as the same as that of MNEI 8 926.52 1077 T1R3 1–25 described by Morini et Morini et al., (2005). Furthermore, 9 924.94 1480 T1R2 115–134 10 922.51 1170 T1R3 119–137 some models of Mab II and Aoc_BA T1R2/T1R3 were also built up from the docking. Among them, a few models 1532 A˚ 2 to be buried at the interface and the extensive showed Mab II can bind to Aoc_BA T1R2 in the way sim- intermolecular interactions being found between Mab II ilar to that of Mab II with Aoc_AB T1R3. However, the and the receptor. These indicate that the model is generally detailed evaluation showed that the contact surfaces in reasonable. Besides, it is notice that the docking areas of both Mab II and Aoc_BA T1R2 are highly positively Mab II with the receptor are roughly in the same site of charged (Fig. 4d), with which the reasonable complex the ‘‘wedge model” as described by Temussi and coworkers model possessing a suitable stereochemistry could not be (Temussi, 2002; Morini et al., 2005). constructed. Therefore, the 3D-docking experiment for Overviewing the representative complex model, the B- the whole models of the receptor hT1R2/T1R3 interacting chain of Mab II is mainly involved in the interaction with with the whole Mabinlin showed that Mab II seems mainly T1R3 (Fig. 4). Looking at the interface of the generated to interact with the T1R3 promoter of Aoc_AB T1R2/ complex three essential surface regions of Mab II, desig- T1R3. nated I1, I2, and I3, respectively, are involved in the direct Combined the observations of the unique structure of contacts with target structure T1R3. I1 mainly consists of Mab II and the docking test of Mab II to hT1R2/T1R3, B-chain segment B54–B69 including the unique [NL/NI] it is plausible to predict that the main structural elements tetralet motif (B54–B64), which is located at the neutral of Mab II for the interaction between Mab II and Face B of the molecule. It is interesting to note that the side hT1R2/T1R3, in turn, for eliciting the sweet taste of Mab chains of the specific four Asn in this motif are just pro- II are in B-chain of Mab II molecule. And the unique motif truded from this surface to touch with the target T1R3, B54–B64 of Mab II might be one of the critical sites for the which implied that the unique [NL/NI] tetralet motif may sweetness of Mab II. These predictions were generally iden- play certain important roles in the interaction of Mab II tified by the following experimental results. with its receptor and elicitation of the sweetness of Mab II. I2 and I3 consist the segments from B-chain’s N-termi- 3.6. Separation of A- and B-chains nus and C-terminus, respectively, which are distributed in the cationic zone and contact to the two partner surface To gain the individual A- and B-chains, the denatured of T1R3 in charge complimentary (Fig. 4c). Mab II protein by 6 M guanidine hydrochloride and 58 D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62

Fig. 4. Mab II-hT1R2/T1R3 complex model constructed by 3D-docking. (a) Overview of the complex structure of Mab II and Aoc_AB T1R2/T1R3 model drawing with a-carbon traces showing that only B-chain of Mab II and the protomer T1R3 of the receptor are involved in contacts. The A- and B- chains of Mab II and T1R2 and T1R3 of the receptor are colored in blue, red, yellow, and green, respectively. (b) Space-ﬁlling drawing of Mab II- T1R3(Aoc_AB) complex showing that the B-chain of Mab II is main structural element involved in contacts with T1R2/T1R3. The A- and B-chains of Mab II and the protomer T1R3 are colored in blue, red, and gray, respectively. (c) Separately drawings of the electrostatic potential surfaces of Mab II (right) and T1R3 (left) in the complex of Mab II and Aoc_AB T1R2/T1R3 model. The three areas involved in the interactions between Mab II and T1R2/ T1R3 are highlighted with circles. The contact site I1 containing the B54–B64 motif of Mab II is located on the neutral Face B showing in Fig. 3. (d) Drawings of the electrostatic potential surface of T1R2 from Aoc_BA T1R2/T1R3 model with the same orientation as in (c), showing the positively charged surface should repel the Mab II’s binding due to Mab II is mainly positively charged. The ﬁgures were prepared by Molscript and PyMol. D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62 59

5mMb-mercaptoethanol was isolated by an AKTA puri- lyophilized samples collected from the elution were used fier system on a C18-reverse-phase column. There were in MALDI-TOF mass spectrum analysis. The resulted two sharp peaks appeared in elution (Fig. 5a). The two molecular mass of Peak 1 and Peak 2 are 4146.50 and 8298.84 Da, which are, respectively, coincident with the calculated masses of A-chain (4168.6 Da) and B-chain (8268.7 Da). The result confirmed that A-chain and B- chain of Mab II have well been separated and purified. Then the fluorescent emission of TrpB72, the only tryptophan residue in the B-chain, was employed to probe the conformational state of the separated B-chain. The wavelength of B-chain’s fluorescence was 352.2 nm which generally coincident with that of the native Mab II (355.6 nm) (Fig. 5b). It has reported that the wavelength of the fluorescence emission from the residue TrpB72 has a significant red shift, from 352 to 370 nm, when Mab II was denatured (Hu et al., 1985a,b). At the same time, 1D 1H NMR spectrum of Mab-B has well dispersed peaks indicating that Mab-B was well refolded and has secondary and tertiary structures (Fig. 5c). These results indicate that the separated Mab-B should have a definite fold and may be adopted a conformation similar to that in the native Mab II.

3.7. Experimental test of interactions between hT1R2/T1R3 and Mab II, Mab-A, Mab-B

To determine whether Mab II, Mab-A, and Mab-B can interact with the human sweet receptor hT1R2/ T1R3, the human sweet receptor was expressed by transient transfection in HEK293E cells along with G16-gust44 and then monitored activation (calcium mobilization) by indicator dye using D-tryptophan as a positive control. The stimulation experiments showed that the human sweet receptor responds to both Mab II (0.5%) and Mab-B (0.5%) and D-tryptophan (10 mM), but essentially not to Mab-A (0.05%), as shown in Fig. 6. The results indicate that both the integrated Mab II and the individual Mab-B can interact with the sweet receptor hT1R2/T1R3 almost in the same scale. It is identiﬁed that Mab-B is the principal subunit of Mab II for its interaction with the sweet receptor hT1R2/T1R3.

3.8. Sweetness evaluation of separated A- and B-chains of Mab II

Fig. 5. Identification of A- and B-chains’ separation. (a) The elution Regular sweetness test was performed by trained peo- profile from a C18 reverse phase column (5 lm, 4.6 250 mm, Buffer A is ple with the regular procedure (Hu and He, 1983). With 0.1% TFA, Buffer B is 0.1% TFA and 70% acetonitrile) on an AKTA the sample solutions at the same concentration both the Purifier system (Amersham Pharmacia Biotech). The molecular masses of Peaks 1 and 2 are determined by MALDI-TOF mass spectrum analysis, native Mab II and separated Mab-B elicited strong which are well coincident with the theoretical values of the A-chain sweetness, but Mab-A did not induce evident sweet feel- (4168.6 Da) and B-chain (8299.7 Da) considering the reasonable error ing. Interestingly, the sweet feelings elicited by Mab II from the facility. (b) The fluorescence emission of Mab II and Mab-B. and Mab-B are rather different. The sweetness produced Compared with the native Mab II the spectrum from the separated B- from Mab-B is immediate and disappears in a short time chain has a small red shift (from 352.2 to 355.6 nm), which indicates that Mab-B is properly refolded as in the native. (c) 1D 1H NMR spectrum of when Mab-B solution is dropped to the tongue of peo- Mab-B. The well-dispersed peaks indicate that Mab-B was well refolded ple, whereas the sweet taste from the native Mab II will and has secondary and tertiary structures. be lasted for half an hour. These results are coincident 60 D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62

4. Conclusion remark

The sweet protein Mab II isolated from the plant of C. masaikai Levl. (mabinlang) consists of two polypeptide chains. The crystal structure of Mab II has been determined at 1.7 A˚ resolution, which is unique in an ‘‘all alpha” fold type to be distinct from all known sweet protein structures. The Mab II shows an amphiphilic molecular surface, in which most of 18 positively charged residue (all Arg) are distributed on the same side of the exterior surface to form a cationic face (Face A), and some polar residue (mainly Asn and Gln) with certain hydrophobic residues are gathered onto another side to create a neutral face (Face B). This unique ‘‘amphipathic design” of the molecule may relate to the primary interactions between Mab II and its receptor. A unique structural motif consisting of the B-chain segment B54–B64 with a special sequence NL–P–NI–C–NI–P–NI is found on the neutral Face B. This motif features four [Asn- Leu/Ile] units connected by three conformation-constrained residues (two Pro and one Cys involved in disulfide bridge), thus called the [NL/I] tetralet motif. In the refined structure, four Asn residues of the motif are rooted in the (Leu/Ile) hydrophobic cluster to be well stabilized and their orientation are directed by Pro or Cys in the disulfide bridge to form a tertiary structural unity on the surface. This special structural organization implies the certain structure–function significance. Following the account of the ‘‘wedge model” for sweet proteins interacting with the sweet-taste receptor (Temussi, 2002; Morini et al., 2005), the 3D-dockiing between Mab II and the sweet receptor hT1R2/T1R3 model were performed, which suggest that the B-chain of Mab II is the main structural element for the interactions with the receptor Fig. 6. Response test of the sweet receptor hT1R2/T1R3 to Mab II, and the unique tetralet motif consisting of residues B54– separated A-chain (Mab-A) and B-chain (Mab-B). HEK-293E cells B64 may be one of the essential binding sites. To experi- coexpressing human T1R2, human T1R3 along with Ga16-gust44 were mentally verify the prediction from the 3D-docking, the loaded with calcium indicator dye Fluo-4, and stimulated with purified two polypeptide chains, A and B, are separated and iden- Mab II (0.5%), Mab-A (0.05%), Mab-B (0.5%), and D-tryptophan (10 mM). Left panels show the fluorescence images of cells before the tified to possess the fold state similar to that in the native addition of the stimuli, and right panels show the fluorescence images 25– Mab II. By using the calcium imaging with the HEK293E 35 s after addition of stimuli. The evident responses to Mab II, Mab-B, cells coexpressed hT1R2/T1R3, the experiments for test- and D-tryptophan, a positive control, were detected, but no response to ing the possible interactions of A-chain, B-chain and the Mab-A was detected. The mock transfected cells show no response to any of them (data not shown). native Mab II, show that hT1R2/T1R3 responds to both the integrated Mab II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the with the observations from the binding test in the cal- sweetness alone, but A-chain does not. However, com- cium imaging experiments described above in which pared with the native Mab II, the special aftertaste is lost hT1R2/T1R3 responded to Mab-B more quickly than it for the individual B-chain. In combination of all data we does to Mab II (data not shown). These tests identified conclude that the sweet protein Mab II can interact with that the B-chain of Mab II can elicit the sweet taste the human sweet-taste receptor hT1R2/T1R3 to elicit its alone and should be the main structural element of the sweet taste. The B-chain with a unique [NL/I] tetralet sweetness elicited by Mab II, while the A-chain may play motif is the essential structural element for the interaction a role in gaining the long aftertaste for the native Mab with the sweet receptor to elicit the sweetness, and the II. The dissection of the main structural basis of the A-chain may play a role in assisting the integrate Mab sweet properties eliciting by Mab II will be of benefit II to gain a long aftertaste. The findings reported in this to develop a unique sweetener from Mab II. paper will be advantage for understanding the diversity D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62 61 of sweet proteins and engineering research for develop- Hu, Z., Peng, L.P., He, M., 1985b. Study on mabinlin, a sweet protein ment of a unique sweetener based on Mab II structure from the seeds of C. masaikai Levl. II: comparison of mabinlin I to as a native model. mabinlin II. Acta Bot. Yunanica 7, 1–10. Jiang, P., Cui, M., Zhao, B., Snyder, L.A., Benard, L.M., Osman, R., Max, M., Margolskee, R.F., 2005. Identification of the cyclamate Protein data bank accession code interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3. J. Biol. Chem. 280, 34296– 34305. Coordinates and structure factors for the structure of Jiang, P., Ji, Q., Liu, Z., Snyder, L.A., Benard, L.M., Margolskee, R.F., Mab II have been deposited at the Protein Data Bank with Max, M., 2004. The cysteine-rich region of T1R3 determines responses accession code 2DS2. to intensely sweet proteins. J. Biol. Chem. 279, 45068–45075. Jin, Z.Y., Danilova, V., Assadi-Porter, F.M., Aceti, D.J., Markley, J.L., Hellekant, G., 2003a. Critical regions for the sweetness of brazzein. Acknowledgments FEBS Lett. 544, 33–37. Jin, Z.Y., Danilova, V., Assadi-Porter, F.M., Markley, J.L., Hellekant, This work was supported by the National Basic G., 2003b. Monkey electrophysiological and human psychophysical Research Program (2006CB806502 and 2006CB911003). responses to mutants of the sweet protein brazzein: delineating We thank Drs. Y. Han and Y. Feng for their helps with brazzein sweetness. Chem. Senses 28, 491–498. 1 Jones, T.A., Zou, J.Y., Cowan, S.W., Kjeldgaard, M., 1991. Improved in-house X-ray data collections and 1D H NMR experi- methods for building protein models in electron density maps and the ments. The authors are also graceful the Photon Factory, location of errors in these models. Acta Crystallogr. A 47, 110–119. KEK (06G191) at Tsucuba and BSRF at Beijing for their Kanarek, R.B., 1994. Does sucrose or aspartame cause hyperactivity in supporting in provison of the synchrotron radiation facili- children? Nutr. Rev. 52, 173–175. ties for the X-ray diffraction experiment. Kaneko, R., Kitabatake, N., 2001. Structure–sweetness relationship in thaumatin: importance of lysine residues. Chem. Senses 26, 167–177. Kant, R., 2005. Sweet proteins—potential replacement for artificial low References calorie sweeteners. Nutr. Rev., 4–5. Kelley, L.A., MacCallum, R.M., Sternberg, M.J.E., 2000. Enhanced Assadi-Porter, F.M., Aceti, D.J., Markley, J.L., 2000. Sweetness determi- genome annotation using structural profiles in the program 3D-PSSM. nant sites of brazzein, a small, heat-stable, sweet-tasting protein. Arch. J. Mol. Biol. 299, 499–520. Biochem. Biophys. 376, 259–265. Kunishima, N., Shimada, Y., Tsuji, Y., Sato, T., Yamamoto, M., Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Kumasaka, T., Nakanishi, S., Jingami, H., Morikawa, K., 2000. Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, Structural basis of glutamate recognition by a dimeric metabotropic N.S., Read, R.J., Rice, L.M., Simonson, T., Warren, G.L., 1998. glutamate receptor. Nature 407, 971–977. Crystallography & NMR system: a new software suite for macromo- Laskowski, R.A., 2001. PDBsum: summaries and analyses of PDB lecular structure determination. Acta Crystallogr. D 54, 905–921. structures. Nucleic Acids Res. 29, 221–222. Caldwell, J.E., Abildgaard, F., Dzakula, Z., Ding, M., Hellekant, G., Lawrence, M.C., Colman, P.M., 1993. Shape complementarity at protein/ Markley, J.L., 1998. Solution structure of the thermostable sweet- protein interfaces. J. Mol. Biol. 234, 946–950. tasting protein brazzein. Nat. Struct. Biol. 5, 427–431. Li, D.F., Zhu, D.Y., Hu, Z., Wang, D.C., 2006. Crystallization and Chothia, 1975. Structural invariants in protein folding. Nature 254, 304– preliminary X-ray analysis of the highly thermostable sweet protein 308. mabinlin II. Protein Peptide Lett. 12, 319–321. Cohe, S., 2001. What’s the truth about the health risks of sugar substitutes Liu, X.Z., Maeda, S., Hu, Z., Toshihiro, A., Nakaya, K., Kurihara, Y., such as saccharin and aspartame? Health News 7, 10. 1993. Purification, comlete amino acid sequence and structural Collaborative Computational Project Number 4 (1994). The CCP4 suite: characterization of the heat-stable sweet protein, mabinlin II. Eur. J. programs for protein crystallography. Acta Crystallogr. D 50, 760–763. Biochem. 211, 281–287. Dauter, Z., Dauter, M., Rajashankar, K.R., 2000. Novel approach to Li, X.D., Staszewski, L., Xu, H., Durick, K., Zoller, M., Adler, E., 2002. phasing proteins: derivatization by short cryo-soaking with halides. Human receptors for sweet and umami tastes. PNAS 99, 4692–4696. Acta Crystallogr. Sect. D 56, 232–237. Mizukoshi, T., Kohmura, M., Suzuki, E., Ariyoshi, Y., 1997. Structure De La Fortelle, E., Bricogne, G. (1997). Maximum-likelihood heavy-atom and dynamic studies by NMR of the potent sweet protein monellin and parameter refinement in the MIR and MAD methods. Methods in a non-sweet analog. Evidence on the importance of residue AspB7 for Enzymology, Macromolecular Crystallography. Academic Press, New sweet taste. FEBS Lett. 413, 409–416. York, 276, 472–494. Morris, J.A., Cagan, R.H., 1972. Purification of monellin, the sweet Ding, M., Hellekant, G., 1994. Brazzein, a new high-potency thermostable principle of Dioscoreophyllum cumminsii. Biochim. Biophys. Acta 261, sweet protein from Pentadiplandra brazzeana B. FEBS Lett. 355, 106– 114–122. 108. Morini, G., Bassoli, A., Temussi, P.A., 2005. From small sweeteners to Ding, M., Hu, Z., 1986. Study on mabinlin, a sweet protein from the seeds sweet proteins: anatomy of the binding sites of the human T1R2_T1R3 of C. masaikai Levl. IV: stability and denaturation. Acta Bot. receptor. J. Med. Chem. 48, 5229–5520. Yunanica 8, 181–192. Murzin, A.G., Brenner, S.E., Hubbard, T., Chothia, C., 1995. SCOP: a Hagiwara, A., Fukushima, S., Kitaori, M., Shibata, M., Ito, N., 1984. structural classification of proteins database for the investigation of Effects of three sweeteners on rat urinary bladder carcinogenesis sequences and structures. J. Mol. Biol. 247, 536–540. initiated by N-butyl-N-(4-hydroxybutyl)-nitrosamine. Gann 75, 763– Nabors, L.O., 1988. Saccharin and aspartame: are they safe to consume 768. during pregnancy? J. Rep. Med. 22, 102. Hu, Z., He, M., 1983. Study on mabinlin, a sweet protein from the seeds of Nirasawa, S., Nishino, T., Katahira, M., Uesugi, S., Hu, Z., Kurihara, Y., C. masaikai Levl. I: extraction, purification and certain characteristics. 1994. Structure of heat-stable and unstable homologues of the sweet Acta Bot. Yunanica 5, 207–212. protein mabinlin. Eur. J. Biochem. 223, 989–995. Hu, Z., Liang, H.X., Liu, X.Z., 1985a. Study on mabinlin, a sweet protein Ogata, C.M., Gordon, P.F., De Vos, A.M., Kim, S.-H., 1992. Crystal from the seeds of C. masaikai Levl. III: storage site and state. Acta Bot. structure of a sweet tasting protein thaumatin I, at 1.65 A˚ resolution. J. Yunanica 7, 187–194. Mol. Biol. 228, 893–908. 62 D.-F. Li et al. / Journal of Structural Biology 162 (2008) 50–62

Orengo, C.A., Michie, A.D., Jones, S., Jones, D.T., Swindells, M.B., sequence weighting, position-specific gap penalties and weight matrix Thornton, J.M., 1997. CATH—a hierarchic classification of protein choice. Nucleic Acids Res. 22, 4673–4680. domain structures. Structure 8 (5), 1093–1108. Tovchigrechko, A., Vakser, I.A., 2005. Development and testing of an Schneider, T., Sheldrick, G., 2002. Substructure solution with SHELXD. automated approach to protein docking. Proteins 60, 296–301. Acta Crystallogr. D58, 1772–1779. Vakser, I.A., 1995. Protein docking for low-resolution structures. Protein Shimizu-Ibuka, A., Morita, Y., Terada, T., Asakura, T., Nakajima, K., Eng. 8, 371–377. Iwata, S., Misaka, T., Sorimachi, H., Arai, S., Abe, K., 2006. Crystal Vakser, I.A., Matar, O.G., Lam, C.F., 1999. A systematic study of low- structure of neoculin: insights into its sweetness and taste-modifying resolution recognition in protein–protein complexes. Proc. Natl. Acad. activity. J. Mol. Biol. 359, 148–158. Sci. USA 96, 8477–8482. Shirasuka, Y., Nakajima, K., Asakura, T., Yamashita, H., Yamamoto, Van der Weld, H., Loeve, K., 1972. Isolation and characterization of A., Hata, S., 2004. Neoculin as a new taste-modifying protein thaumatin I and II, the sweet-tasting proteins from Thaumatococcus occurring in the fruit of Curculigo latifolia. Biosci. Biotechnol. daniellii Benth. Eur. J. Biochem. 31, 221–225. Biochem. 68, 1403–1407. Walters, D.E., Hellekant, G., 2006. Interactions of the sweet protein Somoza, J.R., Cho, J.M., Kim, S.H., 1995. The taste-active regions of Brazzein with the sweet taste receptor. J. Agric. Food Chem. 54, monellin, a potently sweet protein. Chem. Senses 20, 61–68. 10129–10133. Somoza, J.R., Jiang, F., Tong, L., Kang, C.-H., Chao, J.M., Kim, S.-H., Weihrauch, M.R., Diehl, V., Bohlen, H., 2002. Artificial sweeteners— 1993. Two crystal structures of a potently sweet protein. Natural are they potentially carcinogenic? Med. Klin. (Munich) 96, 670– monellin at 2.75 A˚ resolution and single-chain monellin at 1.7 A˚ 675. resolution. J. Mol. Biol. 234, 390–404. Wishart, D., Willard, L., Ranjan, A., Zhang, H., Monzavi, H., Boyko, R., Suzuki, M., Kurimoto, E., Nirasawa, S., Masuda, Y., Hori, K., Kurihara, Sykes, B., 2003. Program Vadar, Faculty of Pharmacy University of Y., 2004. Recombinant curculin heterodimer exhibits taste-modifying Alberta, Edmonton, Canada. and sweet-tasting activities. FEBS Lett. 573, 135–138. Yamashita, H., Theerasilp, S., Aiuchi, T., Makaya, K., Nakamura, Y., Temussi, P.A., 2002. Why are sweet proteins sweet? Interaction of 1990. Purification and complete amino acid sequence of a new type of brazzein, monellin and thaumatin with the T1R2-T1R3 receptor. sweet protein taste-modifying activity, curculin. J. Biol. Chem. 265, FEBS Lett. 526, 1–4. 15770–15775. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. ClustalW: improving Zhang, Y., 2004. The patients of diabetes mellitus have been 50 millions the sensitivity of progressive multiple sequence alignment through populations in China. People Daily 11, 9.