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Plant Science 195 (2012) 135– 142

Contents lists available at SciVerse ScienceDirect

Plant Science

jo u rn al hom epa ge: www.elsev ier .com/ locate /p lantsc i

eview

issimilar sweet proteins from plants: Oddities or normal components?

elia Piconea,b, Piero Andrea Temussia,b,∗

Università di Napoli Federico II, via Cinthia 45, Naples 80126, ItalyNational Institute for Medical Research, The Ridgeway, London NW7 1AA, UK

r t i c l e i n f o

rticle history:eceived 22 March 2012eceived in revised form 30 June 2012ccepted 2 July 2012vailable online 6 July 2012

eywords:

a b s t r a c t

The fruits of a few tropical plants contain intensely sweet proteins. Their common property points to aprotein family. Generally, proteins belonging to the same family share similar folds, similar sequencesand, at least in part, similar function but sweet proteins constitute an exception to this rule. Apart fromsharing the rather unusual taste function, they show no obvious similarities either in their sequences orin three-dimensional structures.

In this review we describe the nature, structure and mechanism of action of the best known sweet tast-

astenhibitorsropical plantsweet taste receptorodelling

ing proteins, including two taste modifying proteins. Sweet proteins stand out among sweet moleculesbecause their volume is not compatible with an interaction with orthosteric active sites of the sweet tastereceptor. The best explanation of their mechanism of action is the interaction with the external surfaceof the sweet taste receptor, according to a model that has been named “wedge model”. It is hypothesizedthat this mode of action may be related to the ability of other members of their protein families to inhibitdifferent enzymes.

© 2012 Elsevier Ireland Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1352. Sweet proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

2.1. Plant origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1362.2. Architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1362.3. Family profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

3. Structural determinants of sweet proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1373.1. Intensely sweet proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1373.2. Taste modifying proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

4. Proteins as sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1405. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

. Introduction

Most people associate sweet taste with sugars or some non-aloric sweetener, but it is very uncommon in everyday life tossociate sweet taste with proteins. However, sweet proteins

in unrelated tropical plants and, apart from the tropical plantorigin, have very little in common: when considered as a family,sweet proteins show no functional similarity among them eitherin sequence or in three-dimensional structure. They interactwith the same receptor that binds small sweet molecules, but

o exist and are sweeter than sucrose and most non-caloricweeteners. Some of them are indeed intensely sweet, orders ofagnitude sweeter than sugar [1]. Therefore their sheer existence

as always posed several puzzling problems. They are all found

∗ Corresponding author at: Dipartimento di Scienze Chimiche, via Cinthia 45,aples 80126, Italy. Tel.: +39 081674416; fax: +39 081674409.

E-mail address: temussi@unina.it (P.A. Temussi).

168-9452/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.plantsci.2012.07.001

their sheer volume prevents them from entering the orthostericactive sites that can host small molecular weight sweeteners. Forinstance, the molecular volume of aspartame can be estimatedas 270 A3 (http://www.molinspiration.com/cgibin/properties)whereas the molecular volume of monellin, is about 13,400 A3

(http://www.basic.northwestern.edu/biotools/proteincalc.html).Thaumatin, monellin and brazzein, are considered the prototyp-

ical sweet proteins, both from structural and functional points ofview [1–3]. Among the sweet proteins of plant origin there is also

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abinlin, which however, is far less sweet than the three proteinsentioned above [2]. Strictly speaking it is not possible to classify

s sweet proteins miraculin [2] and curculin [4]. They have little oro sweet taste but have the property of changing sour into sweetaste. Recently egg white lysozyme has also been proposed as a

ember of the family [5], but, in addition to its animal origin, itsuch lower sweetening power puts it into a different class with

espect to sweet proteins of plant origin. Accordingly, this reviewill deal only with monellin, thaumatin, brazzein and mabinlin

s intensely sweet proteins and miraculin and neoculin as tasteodifying proteins.

. Sweet proteins

All known distinctly sweet proteins were discovered in tropi-al plants. However, no correlation apparently exists among theselants, apart perhaps between Pentadiplandra brazzeana, the plantroducing brazzein, and Capparis masaikai Levl., the plant produc-

ng mabinlin, because they belong to the same order (Brassicales).

.1. Plant origin

Monellin was discovered in Dioscoreophyllum cumminsii, aigher altitude variant of Dioscoreophyllum volkensii, a tropical rain-

orest vine in the family Menispermaceae [6]. The plant, also knowns serendipity berry, is native in most countries of tropical Africa. Itselative molar sweetness, with respect to sucrose, is approximately00,000:1 [1].

Thaumatin was found in the aril of the seeds of Thaumatococcusanielli, a large flowering herb native to the rainforests of Ghanand surrounding African nations [6]. The sweet substance is pro-einaceous [7], a mixture of thaumatins I and II [8]. Both proteins,ike monellin, are approximately 100,000 times sweeter than sugar1].

P. brazzeana, the sole species in its genus (family Pentadiplan-raceae), grows in Angola, the Democratic Republic of Congo andther countries in tropical Africa. The interest in its sweet princi-le was aroused when the plant, known as Oubli in Gabon, was,o to speak, rediscovered in 1985 by researchers studying the eat-ng habits of apes. Brazzein has been isolated more recently thanhaumatin and monellin [3]. Pentadin is sometimes quoted amongnown sweet proteins as another sweet protein from the samelant, but it is now established that pentadin and brazzein are justwo names of the same protein (G. Hellekant, personal communi-ation). Brazzein, although intensely sweet, is less powerful thanonellin and thaumatin [3].Mabinlang is the Chinese name of C. masaikai Levl. the plant

here mabinlin is found [9]. The two proteins responsible for theweet taste of Capparis masaikai Levl. were dubbed mabinlin I andI. All subsequent studies have been done on mabinlin II, which is75 times sweeter than sugar [10]. Thus, although it is undoubtedlyweeter than sucrose, it ought to be placed in a different class fromhe three mentioned prototypical sweet proteins whose sweeten-ng power is on average two orders of magnitude higher. MabinlinI has little similarity to other sweet proteins [10]. However, theequence of mabinlin II is similar to that of 2S albumin AT2S3 ofrabidopsis thaliana [11], which is not sweet.

Miraculin is not sweet by itself, but can change the taste ofour substances into sweet. The fruits of Synepalum dulcificumalso called Richadella dulcifica), from which miraculin is isolated,ave been called ‘miracle fruit’ owing to the surprising property of

hanging sour substances into sweet ones [12,13].

The fruits of Curculigo latifolia, a plant from Malaysia containurculin, another taste-modifying protein. Curculin itself elicits

sweet taste, but early reports [14,15] are somewhat confusing

ence 195 (2012) 135– 142

because they are at variance with later findings on neoculin, nowregarded as the authentic active form of curculin [16]. The rela-tive sweetness is equivalent to that of a 0.35 M solution of sucroseat the concentration corresponding to saturation in the mouth(10 �M), which would correspond to a relative sweetness of 35,000times than sucrose [15]. However, the same researchers [15] reportexactly the same value for sweetness induced by sour substances. Ifthese data [14,15] were taken at face value, curculin should be clas-sified as an intensely sweet protein, in the same class of monellinthaumatin and brazzein. However, subsequently it was demon-strated that curculin (called by them curculin1) is only one of thetwo isoforms and that it is necessary to combine it with a new iso-form (curculin2) to produce the bona fide heterodimer that has apowerful taste modifying property [17]. The heterodimer, dubbedneoculin at neutral pH is only slight sweet but becomes very sweetat acidic pH [16].

2.2. Architecture

Sweet proteins have sequences of widely different length andvirtually no homology. They have also different 3D folds, albeitmostly of the alpha–beta type, with the only exception of mabin-lin that is all alpha, as found in the Protein Data Bank (pdb,http://www.rcsb.org).

Monellin consists of two separate peptide chains forming asingle domain through non-covalent interactions [18]. It has analpha–beta architecture with a cystatin-like fold, where the sin-gle helix is cradled in a coiled antiparallel beta-sheet [19] andhas been classified as a member of the cystatin superfamily [20],although devoid of protease inhibitory function. Monellin losessweetness when heated above 50 ◦C, a clear indication that thewhole three-dimensional structure is a prerequisite for sweet-ness, because at high temperature the two chains held togetherby non-covalent forces dissociate. Single chain constructs (Fig. 1a),obtained by joining the two chains either by a direct amide bondor by the insertion of a dipeptide, are as sweet as native mon-ellin, and as an additional bonus have greatly increased thermalstability [21,22]. Both single chain constructs refold easily evenafter boiling at low pH and are as potently sweet as wild typemonellin.

Thaumatins (I and II) have 207 residues, differing only at fourpositions [8]. The 3D structure of thaumatin, a member of theosmotin superfamily [23] has been determined by several differ-ent solid state structural studies, mainly on thaumatin I [24–26],but also on thaumatin II [27]. The large number of solid state stud-ies is motivated, in part, by the fact that this protein is used as abenchmark for crystallization [28]. The 3D structure of thaumatin I(pdb id 3al7, Fig. 1b) contains three domains. The main domainis made of a long beta sheet fold of 11 beta strands [24]. Eachstrand is antiparallel to its neighbours with the exception of thefirst and last strands, which are parallel to each other. Two smalldomains rich in disulfide bonds are attached to this domain. Thestructure of thaumatin II is almost indistinguishable from that ofthaumatin I.

The sequence of brazzein is made of only 54 amino acidresidues. The brazzein architecture is characterized by threestrands of antiparallel beta-sheet and one alpha-helix [29](Fig. 1c). Brazzein owes the stability of its structure to the presenceof the disulfide bridges, like plant defensins and arthropod toxins,with which it shares a similar architecture.

Mabinlin II is characterized by two chains linked by disulfidebridges [10]. The structure of mabinlin II has been solved by X-

ray diffraction [30]. The fold (Fig. 1d) is typical of an all alphaprotein, according to SCOP (Structural Classification Of Proteins)(http://scop.mrc-lmb.cam.ac.uk) [23]. The two chains are linkedby two disulfide bridges. Two other intra chain disulfide bridges

D. Picone, P.A. Temussi / Plant Sci

Fig. 1. Molecular models of the main sweet proteins. (a) Single chain monellin (pdbid: 1fa3). (b) Thaumatin I (pdb id: 3al7). (c) des-pyro Glu brazzein (pdb id: 2KGQ). (d)Mia

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abinlin II (pdb id: 2ds2). (e) Neoculin (pdb id: 2d04). The neoculin acidic subunits the darker ribbon, the neoculin basic subunit is the paler ribbon. Models, showns cartoon ribbons surrounded by dot surfaces, were built with Pymol [75].

re present in the B-chain. The A-chain contains two alpha-helicesonnected by a turn.

The structure of taste-modifying neoculin has been solved by X-ay diffraction [16]. The two chains, linked by two disulfide bridges,ave been called neoculin acidic subunit and neoculin basic sub-nit: They have nearly identical length (113 and 114 residuesespectively) and very similar structures. Each subunit containshree beta-sheets forming an irregular triangular prism (Fig. 1e).

The sequence of taste-modifying miraculin has 191 amino acidesidues. No three-dimensional structure of miraculin has beenublished. Two homology models, albeit not deposited in the PDB,ave been reported [31,32] and there is a structure of a miraculin-

ike protein [33]. This latter protein shares 39% identity withiraculin. The high similarity of the sequence of miraculin with

hose of Kunitz protease inhibitors allows meaningful homologyodelling using structures of these inhibitors as templates [34].odels of miraculin were built as homodimers [31,32]. The dimer,

n which two polypeptide chains of miraculin are linked by disul-de bridges, is the minimum unit that can exert the taste-modifyingroperty [35]. Although in one case [31] the nature of the templateas not mentioned, it is fair to surmise it was a Kunitz protease

nhibitor.

.3. Family profile

Plant fruits, notably in their seeds contain many proteins thatan act in response to pathogenic attack by pests and pathogens,

ence 195 (2012) 135– 142 137

including a variety of protease and other enzyme inhibitors [36].Their transcription is mainly detected in the development and seedgermination phases [37]. These proteins have interesting similari-ties with plant sweet proteins, suggesting in some cases a commongenetic origin.

Recently a new Cys protease inhibitor belonging to the mul-ticystatin family has been identified in the subphellogen layer ofpotato tubers (Solanum tuberosum) [38]. It is composed of eightsuperimposable domains of about 10 kDa with slightly differentprimary structures that are connected by short linkers. A detailedcomparison of the crystal structure with available structures inthe Protein Data Bank revealed a strong similarity with the singlechain monellin, despite the low sequence identity, thus confirmingthe hypothesis that plant cystatins and monellin descend from acommon ancestor [20].

Thaumatin itself gives the name to a protein family [23]. Athaumatin-like xylanase inhibitor (TLXI) has been very recentlydiscovered in wheat (Triticum aestivum) [39]. Like other plantthaumatin-like proteins (TLP), TLXI contains 10 cysteines that formfive internal disulfide bridges. The X-ray structure indicates thepresence of two domains, lacking the region corresponding to thau-matin domain II [40].

According to SCOP, brazzein belongs to the scorpion toxin-likesuperfamily [23]. An A. thaliana trypsin inhibitor, although char-acterized by only two strands of antiparallel beta-sheet and onealpha helix, has architectural similarities with that of brazzein [41].The alignment of the two protein sequences revealed 28% identityand 38% homology, whereas the rmsd calculated between the 3Dstructure (PDB entries 1JXC and 2BRZ) was 3.0 A.

Mabinlin does not belong to a recognizable protein family. How-ever, it is similar to a 10 kDa protein isolated from dormant Cannabissativa seeds: 36 residues are identical, 26 homologous. Originallydeclared a trypsin inhibitor [36], the purified protein has only abarely detectable inhibitory activity. Like mabinlin, this protein hastwo subunits of different size linked by two disulfide bonds. No 3Dstructure of this protein is available. It contains an unusually highpresence of sulphur containing amino acid residues: 8 methioninesand 8 cysteines out of a total of 88 residues.

Based on sequence homology, which ranges from 30 to 95%identity, an entire class of plant protease inhibitors is commonlyidentified as “miraculin-like proteins”. Several “miraculin-like”proteins have been structurally characterized [42], however theirsweetness or taste modifying activities have not been investigated.Unfortunately there are no direct structural data on miraculinto evaluate the similarity of 3D structures, but the high degreeof homology with miraculin led to proposals that miraculin,miraculin-like proteins and soybean trypsin inhibitor have evolvedfrom a common ancestor [43].

There are no structural similarities among plant defence pro-teins and curculin/neoculin nor obvious similarities with proteaseinhibitors. However, there is sequence similarity between curculinand tarin [44], the storage protein of taro (Colocasia esculenta L.Schott), which has trypsin inhibitor activity [45]. The neoculin sub-units have a high degree of sequence homology with some mannosebinding lectins (42–46% identity) [46]. There are no functional linksbetween neoculin/curculin and mannose-binding lectins [16]. Themain features of sweet proteins are summarized in Table 1.

3. Structural determinants of sweet proteins

3.1. Intensely sweet proteins

The oral sweet taste receptor is a G protein coupled receptorof class C. In addition to the seven helix transmembrane domaincharacteristic of this family of receptors, it also has a large extra

138 D. Picone, P.A. Temussi / Plant Science 195 (2012) 135– 142

Table 1Summary of the properties of sweet proteins of plant origin.

Protein Res. Class (family) Active form Plant Relative Sweetness (molar,referred to sucrose)

Ref.

Brazzein 54 �/� (scorpion toxin-like superfamily)a Monomer Pentadiplandra brazzeana B. 17,000 [3]Monellin 94 �/� (cystatin-like)a Heterodimer or monomerb Dioscoreophyllum cumminsii 100,000 [6]Thaumatin 207 All �a (thaumatin-like) Monomer Thaumatococcus danielli 100,000 [6]Mabinlin 105 All �a Heterodimer Capparis masaikai Levl. ≈400 [9]Miraculin 191 �/� Homodimerc Synepalum dulcificum None [13]Neoculin 227 All � Heterodimer Curculigo latifolia ≈0.35 M sucrose [16]

a SCOP classification [23].

mddthVratspo

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on a large external site of the active conformation (Fig. 2c). Dock-ing calculations, performed on monellin, thaumatin, and brazzein,showed that in all three cases the protein molecule bind to the

Fig. 2. The wedge model. (a) Representation of the equilibrium between the freeform I (dubbed FF I in this figure) and the free form II (dubbed FF II in this figure)conformations of the sweet receptor. At rest the equilibrium is shifted in favour ofform FF I. (b) Representation of the equilibrium between the resting conformation

b Single chain derivatives have been obtained by protein engineering.c Based on modelling studies.

embrane domain, called Venus flytrap domain and a cysteine-richomain connecting the previous two domains. The Venus flytrapomain, named after the trap closure of a carnivorous plant, con-ains an active site for typical orthosteric ligands similar to thoseosted by other class C G protein coupled receptors. When theenus flytrap domain “eats” a ligand, it closes in a manner thatesembles the analogous change of shape of the plant after it eatsn insect. Contrary to most other class C G protein coupled recep-ors, which function as homodimers, the functional form of theweet receptor is that of a heterodimer. The sequences of the tworotomers, dubbed T1R2 and T1R3 by geneticists, are highly homol-gous but not identical [47].

The homology of the sequences of the T1R2 and T1R3 pro-omers of the sweet receptor with that of a metabotropic glutamateeceptor, called mGluR1, has allowed homology modelling bothor T1R3 homodimers [48,49] and for the T1R2–T1R3 heterodimer50]. There are multiple solutions for the dimer because the Venusytrap domain of the mentioned metabotropic glutamate recep-or, even in the absence of ligands, can exist as a mixture of twoonformations in equilibrium [51]. Threading the two sequencesf T1R2 and T1R3 into the two conformations of the metabotropiclutamate receptor, gives rise to four possible heterodimers: anctive open–closed one (using pdb id: 1ewk and 1ewv as templatesespectively) and an inactive open–open free form I (using pdbd: 1ewt as template). The models of active sites of the Venus fly-rap domains of the sweet receptor explain the taste of most small

olecular weight sweet molecules and the phenomenon of syn-rgy but cannot be used in any obvious way to explain the activity ofweet proteins, as illustrated in a detailed refinement of the “wedgeodel” [52]. This paper contains for the first time the explicit pro-

osal that the sweet taste receptor hosts multiple binding sites52], an idea since accepted by most other groups (see for instance53]).

The common function of the members of a protein family canenerally be accounted for on the basis of sequence homology or ofimilar 3D structures. As already stated above, sweet proteins doot have any sequence homology [54], nor any obvious similaritymong their folds and all attempts to identify sweet fingers in smallrotruding structural elements failed [54]. Mutations affecting theweetness of monellin [55] and brazzein [56] are not restricted tomall regions but spread over a large surface. This indication, com-ng from mutagenesis studies, is consistent with the observationhat most, if not all, sweet proteins belong to families in whichunction requires interaction with a large area of another protein,otably in the case of protease inhibitors, as described above. Inur view, the best model that can interpret the action of all sweetroteins is the “wedge model” [1,50,52]. It accounts explicitly forhe need of involving large surface areas of sweet proteins and a

orresponding large surface of the sweet receptor.

This model is based on the high homology with mGluR1, whicht the time of the discovery of taste receptors was the only class C Grotein receptor of known structure [51]. This glutamate receptor,

even in the absence of any ligand, exists as a mixture of two confor-mations in equilibrium, an inactive form and another conformationidentical to the active conformation containing the ligand. In thewedge model it was assumed that a similar equilibrium exists alsoin the case of the sweet receptor. The two conformations, in thefield of class C G protein coupled receptors are called Roo, i.e. restiveopen–open and Aoc, i.e. active open–closed, with reference to theconformations of the two Venus flytrap domains that can be openor closed, like the mouth of the carnivorous plant. In the top panelof Fig. 2a it is shown that the inactive free form (dubbed FF I in thefigure) exists in equilibrium with a smaller population of free formII (dubbed FF II).

Addition of small molecular weight sweeteners, which canoccupy the orthosteric cavities of the Venus flytrap domains, shiftsthe equilibrium between the two conformations in favour of theactive form (Fig. 2b). In the wedge model [50], it was shown that theequilibrium can also be shifted by the binding of a macromolecule

of the sweet receptor (dubbed Roo in receptor literature, left) and the active confor-mation of the sweet receptor (dubbed Aoc in receptor literature, right), stabilized bya small molecular weight sweetener (ball on Aoc). (c) Representation of equilibriumbetween Roo and Aoc, stabilized by a sweet protein (represented as a dark wedge),binding to an external cavity involving both protomers (T1R2 and T1R3).

D. Picone, P.A. Temussi / Plant Science 195 (2012) 135– 142 139

Fig. 3. Topologically correct model of a single chain monellin with the sweet receptor. (a) The free form II (identical to the active conformation) is converted by a sweetprotein (a single chain monellin, represented as a purple wedge) into the active Aoc form (b). The molecular model of the sweet taste receptor is represented as a contactsurface, with a linear fog effect to enhance the perception of depth. Both protomers are green: T1R2 is pale green, T1R3 is dark green. (c) Interacting residues on the receptori the rek ith brg e ellip

apgdlrt

wssdfsfaistv

nterface responding to a single chain monellin and brazzein. The two protomers ofey residues of a single chain monellin are coloured in purple, those interacting wold. The area hosting residues interacting with the two sweet proteins is within th

ctive conformation of the receptor as a ‘wedge’ [50,52]. The topanel of Fig. 3a and b illustrates this binding in the case of a sin-le chain monellin, using molecular models of the Venus flytrapomain of the receptor and of the single chain monellin. Stabi-

ization of the complex between the active conformation of theeceptor and monellin is due to the large interacting surfaces ofhe proteins.

The mechanism of action of mabinlin can be explained by theedge model [30]. However, the low resolution docking repre-

ents in this case just a proof of concept, albeit interpreted byome as accurate structure determination [55]. The resulting three-imensional models of the complexes with the receptor are auzzy representation of the complexes, because of their intrin-ic low resolution. The best wedge model complex of brazzein,ound via an ingenious scoring method but without taking properccount of the multiple nature of the solution [57], was found

nconsistent with new mutations [56]. The reason for this incon-istency, was due to the intrinsic difficulty of the blind use ofhe model [58]. In the ensemble of a given sweet protein, indi-idual protein molecules can bind with different orientations of

ceptor are coloured in green as above. The side chains of residues interacting withazzein are coloured in red, D456 A that is common to both surfaces is coloured inse. Models were built with MOLMOL [76].

the same surface or even with different parts of their surface[58]. Selection of single meaningful complexes from the ensem-bles requires either the experimental determination of a highresolution structure or the identification, by independent tech-niques, of key residues on sweet proteins and/or on the receptor,which may define the surface of interaction.It is indeed possi-ble to yoke high resolution structures of sweet proteins to lowresolution models of the sweet receptor by finding accurate loca-tions of charged residues [58]. The use of accurate coordinatesfrom key (charged) residues on the sweet protein imposes strongtopological constraints because it is very unlikely that severalsweet protein residues have a one to one correspondence toreceptor residues only by chance. The topologically correct mod-els of the complexes of monellin and brazzein with the sweetreceptor are consistent with the distribution of charged residuesand explain data not used in their derivation [58]. In particular,

the new complex of monellin is fully consistent with a previ-ously reported supersweet MNEI [59]. Likewise, the effect of mostmutated residues in brazzein can be explained by interactions withthe receptor. The most interesting aspect of the model derived

1 nt Sci

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ere for brazzein is the possibility of rationalizing two of theost surprising mutations, namely the maximal increase of sweet-

ess obtained with either the D40K mutation or with the doubleutant E41K/K42E [56]. Brazzein and monellin have different

equences and structures [58]. Do they interact with the sameesidues of the receptor? We can have an immediate answer byooking at Fig. 3c. Residues interacting with two key sweet pro-ein residues are plotted on the Aoc form of the sweet receptorith different colours for the two proteins. Receptor residues inter-

cting with MNEI are coloured in purple, those interacting withrazzein are coloured in red, D456 that is common to both sur-aces is coloured in gold. The nearly perfect superposition of thewo areas speaks highly in favour of the validity of the wedge

odel [58].While the wedge model is the best known mechanism that

an explain the puzzling phenomenon of sweet proteins, it is notnique. Another model was proposed to explain the sweet tastef brazzein [60]. This model identified the cysteine rich domain ashe region of the sweet taste receptor that recognizes and bindsweet proteins. Recently, on the basis of experiments similar to theriginal ones [60], the same mechanism has been proposed alsoor thaumatin [61]. Both groups used chimera receptors obtainedy combining human T1R2 with either human or mouse T1R3. Theationale of this approach is that since humans recognize sweetroteins but mice do not, a comparison of the sequences of activend inactive chimeras obtained from human and mouse sequenceshould point to residues absolutely essential for the recognitionf sweet proteins. In so doing some residues of the cysteine richomain are crucial for active recognition of brazzein molecules andossibly useful for the recognition of monellin, albeit the data onhis last protein proved not conclusive [60] determined that. Bothhe original experiments [60] and the most recent ones [61] areawless and hint at the importance of structural integrity of theeceptor for correct functioning. However, the ensuing identifica-ion of the cysteine rich domain as the preferred binding site for allweet proteins is not straightforward. The main difficulty residesn the very structure of the cysteine rich domain. Although thatf the sweet receptor has not yet been determined experimen-ally, it is highly homologous to those of other G protein coupledeceptors of class C whose X-ray structures have been recentlyetermined [62]. It is clear that all cysteine rich domains haveery thin, filament-like structures, as required by their function ofonnecting the Venus flytrap and transmembrane domains, keep-ng them well separated and possibly transducing conformationalhanges from the Venus flytrap domain to the cytoplasmic end ofhe trans membrane domain [62]. As reported above, point muta-ion studies on brazzein have convincingly shown that the residuesrucial for eliciting sweet taste are spread over a large surface ofhe protein [56]. It is very difficult to envisage a physical contactetween the large surface hosting key residues of even the small-st of the sweet proteins, i.e. brazzein, and the filiform cysteineich domain. It would be more difficult to find charged residues onhe surface of the cysteine rich domain that can match key chargedesidues on the surface of brazzein. It is possible, by a judiciousse of the wedge model, to match every single crucial residue ofrazzein and monellin to corresponding residues of opposite chargen the receptor [58]. Such a feat would be impossible in the casef cysteine rich domain, simply because the available surface is notarge enough.

Accordingly, it is not possible to hypothesize the cysteine richomains as a potential binding site. The explanation of the molec-lar biology data [60,61] is simply in the fact that the cysteine rich

omain is essential for a proper transmission of the signal fromxternal binding sites to the TM domains and thus any mutationhat changes the structure of cysteine rich domain can damageroper functioning of the receptor [62].

ence 195 (2012) 135– 142

3.2. Taste modifying proteins

The mechanism by which miraculin and neoculin are able tochange sour taste into sweet taste has puzzled scientists sincethe discovery of miraculin [13]. A plausible mechanism was pro-posed long before the sweet receptor was discovered [63]. Themodel was essentially allosteric, involving the receptor membraneand the receptor itself, whereas no direct influence of low pHon the conformation of miraculin was invoked. Recently differentmechanisms have been proposed for neoculin [16] and miraculin[32,64,65]. These authors favour a conformational change in thetaste-modifying proteins as the major effect of induced by loweringthe pH and hence inducing sweetness.

The starting point for these very similar models is the fact thatneoculin is slightly sweet at neutral pH and very sweet at acidic pH.The change of taste intensity of neoculin as a function of pH washypothesized to be due to a conformational change of the neoculinheterodimer [16]. The key residues for the conformational changewere identified as two histidines (His11 and His14) located on thesurface of the basic protomer. The protonation of these residuesmight start a conformational transition that separates neoculin acidsubunit and the neoculin basic subunit, the two protomers, allow-ing a different interaction of the open dimer with the receptor [16].Likewise, the two His29 residues of the miraculin homodimer mayrepresent a plausible starting point of a conformational change ofthe dimer leading to a more open state [64].

These twin models are certainly very suggestive of plausiblemechanisms for the two taste modifying proteins, but for oneimportant reservation. Miraculin can exert its taste modificationonly at very low pH values, depending also on the proton counte-rion: for instance, only below pH 3.5 for acetic acid and below pH 1.7for citric acid [63]. In the light of these experimental observationsit is seems unlikely that His residues play a major role, becauseit is extremely unlikely that their pKs are as low as 4.5 or evenlower. With a pK around 6.0, a well accepted average value for a Hisresidue, the population of charged His should prevail by an orderof magnitude over the uncharged one already at pH 5.0, a value atwhich no taste modifying ability was observed, at least for mirac-ulin. The pH range in which this activity is observed is typical of thetitration range of carboxylic groups [16,63]. Accordingly, it seemsdesirable to look for different mechanisms, possibly involving alsoreceptor residues.

4. Proteins as sweeteners

Owing to their exceptional activity, sweet proteins have beenconsidered as obvious targets for biotechnology applications,although the actual use of a molecule as an alternative sweet-ener depends on a complex combination of many factors [66].Two reviews cover all research up to 2006 [67,68]. Two mainpaths have been followed: the direct use of purified sweet pro-teins as sweeteners or the expression of their genes in plantscommonly used as food. Availability of the three main very sweetproteins in quantities significant for their use in the food indus-try can be achieved by extraction from the parent tropical plants,by expression in microorganisms or by expression in plants dif-ferent from those from which they originate. Thaumatin can beprepared from the fruits of the tropical plant that produces it ineconomically convenient yields, but neither monellin nor brazzeinhave been extracted in industrial quantities from their tropicalplants [67].

All three proteins have been expressed successfully in sev-eral organisms but in some cases expression of sweet proteinsin microorganisms, although apparently successful, led to prod-ucts devoid of sweetness. It is possible to speculate that, in the

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arent plants their taste is modulated by other components.ynergy is not a new phenomenon in sweet taste; simulta-eous exposure to two ligands can generate a taste sensationtronger than the sum of the two [69]. The presence of two dis-inct sites for orthosteric ligands in the heterodimeric receptoruggests that, although the binding of one ligand is sufficiento activate the receptor, the binding of a ligand in the sec-nd active site can increase the intensity of the taste sensation52]. A similar synergic mechanism is difficult to envisage forweet proteins if it is accepted that they bind externally, accord-ng to the wedge model. Even more important, it is now wellstablished that pure samples of all three proteins and of theirutants have relative sweetness orders of magnitude higher

han sucrose [3,21,24,27,29]. It would be difficult to improvever these extremely high values by any synergistic action. Its likely that the lack of biological activity in some of the earlyxpressions [67] was due to the difficulty of properly refolding arotein with disulfide bonds. This is particularly true for thaumatinnd brazzein that contain several disulfide bonds. Accordingly,t seems unlikely that these extremely sweet proteins need theelp of other molecules to express their function. It is possible toxclude that the intensity of their sweetness is due to the pres-nce of post-translational modifications because sweet proteinsxpressed in Escherichia coli, an organism that does not allowost-translational modifications, have very high relative sweetness19,21,22,25,55].

Engineering sweet proteins into edible plants, different from theriginal tropical ones, seems a very attractive prospect because,f successful, could lead to direct use of such plants, withouthe need of isolating the proteins and might be more accept-ble than expressing them in micro-organisms. Thaumatin haseen expressed in potato, cucumber, tomato, pear and straw-erry [68] but the low yields might have limited the usefulnessf the approach, considering that this protein can be easilyxtracted from the plant. So far, direct use of sweet proteinss food additives has been limited to thaumatin, commercial-zed in UK, after its approval in 1983, by the Tate & Lyle Co.he use in animal feed and pet food is somewhat doubtfulecause some authors claim that only primates do taste sweetroteins [60,70]. However, it has been suggested that thaumatin,outinely added to pig food, may be sensed through non-tasteissues, particularly in the mucosa of the gastro-intestinal tract71]. Monellin and brazzein have not reached the market so far.

few additional papers dealing with the expression of sweetroteins for biotechnological purposes have recently appeared72–74].

. Conclusions

The sweet taste of many plants, or parts thereof, is gener-lly associated with sugars but it is now well known that theruits of some tropical plants, particularly plants from regions thatave fostered the evolution of primates, contain sweet proteins.hey are intensely sweet, several orders of magnitude sweeterhan sugars, but taste sweet only to primates. Besides their pres-nce in tropical plants, they have very little in common, accordingo usual ways of comparing proteins with the same function.hey have very different amino acid sequences and differenthree-dimensional structure. Their evolution from proteins withntirely different function appears to be related to the mutualdvantage (for plants and primates) to spread the seeds. Sweetroteins have been considered, so far, oddities rather than nor-

al components of tropical fruits. Should directed searches of

ntensely sweet tropical plants be promoted it may well turnut that sweet proteins are more widespread than presentlyhought.

[

ence 195 (2012) 135– 142 141

Acknowledgement

We thank Annalisa Pastore (NIMR) for hospitality, constantencouragement and many important suggestions.

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