Interaction of sweet proteins with their receptor - Wiley Online Library

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the carboxyl (AH) and amino (B) groups of Asp and the aromatic ring of Phe in ... a structural entity hosting all essential glucophores, i.e. the mentioned sweet ...
Eur. J. Biochem. 271, 2231–2240 (2004)  FEBS 2004

doi:10.1111/j.1432-1033.2004.04154.x

Interaction of sweet proteins with their receptor A conformational study of peptides corresponding to loops of brazzein, monellin and thaumatin Teodorico Tancredi1, Annalisa Pastore2, Severo Salvadori3, Veronica Esposito4 and Piero A. Temussi2,4,5 1

Istituto di Chimica Biomolecolare del CNR, Pozzuoli, Italy; 2National Institute for Medical Research, Medical Research Council, London, UK; 3Dipartimento di Scienze Farmaceutiche, Universita´ di Ferrara, Ferrara; 4Dipartimento di Chimica, Universita` di Napoli Federico II, Napoli; 5Centro Linceo ‘Beniamino Segre’, Accademia dei Lincei, Rome, Italy

The mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor. The presence on the surface of the proteins of ‘sweet fingers’, i.e. protruding features with chemical groups similar to those of low molecular mass sweeteners that can probe the active site of the receptor, would be consistent with a single mechanism for the two classes of compounds. We have synthesized three cyclic peptides corresponding to the best potential ‘sweet fingers’ of brazzein, monellin and thaumatin, the sweet proteins whose structures are well characterized. NMR data show that all three peptides have a clear tendency, in aqueous solution, to assume hairpin conformations consistent with the conformation of the same

sequences in the parent proteins. The peptide corresponding to the only possible loop of brazzein, c[CFYDEKRNL QC(37–47)], exists in solution in a well ordered hairpin conformation very similar to that of the same sequence in the parent protein. However, none of the peptides has a sweet taste. This finding strongly suggests that sweet proteins recognize a binding site different from the one that binds small molecular mass sweeteners. The data of the present work support an alternative mechanism of interaction, the ‘wedge model’, recently proposed for sweet proteins [Temussi, P. A. (2002) FEBS Lett. 526, 1–3.].

Sweeteners are widely used by people affected by diseases linked to the consumption of carbohydrates, such as diabetes and obesity. Although sugar substitutes are now reasonably safe, there is a continuous search for new, safer ones. Their design requires a good understanding of the interaction of sweet molecules with their receptor. Ideally, one would like to determine the structure of sweet molecules inside the receptor but, although sweet taste receptors have been recently cloned and expressed [1,2], direct structural studies of membrane proteins are very difficult. In order to study the structure– activity relationship of sweet molecules it is possible to recur to indirect investigations. The great majority of sweet molecules are small molecular mass compounds [3] but a few sweet proteins have also been identified [4]. Low molecular mass sweet compounds and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor [5]; accordingly, it seems natural to look for a similar mechanism when trying to explain the taste of these proteins. Early models of the active site of the sweet receptor, proposed before the nature of the receptor was elucidated, were derived from the shape of conformationally rigid

sweeteners, used as molecular molds [6–12]. Different models differ in details of the shape of the active site but there is a consensus on the presence, in the sweetener, of two groups involved in hydrogen bonding with the receptor, the so-called AH-B entity [6], and of an apolar group at a precise distance with respect to the AH-B entity [7]. A well known exemplification of these three groups is provided by the carboxyl (AH) and amino (B) groups of Asp and the aromatic ring of Phe in aspartame [9]. Although designed for small molecules, these models could be compatible also with the interaction of macromolecular sweeteners with the receptor, provided sweet macromolecules possess a ‘sweet finger’ that can probe the active site. Such a protruding feature should contain glucophores, i.e. key groups responsible for the biological activity, similar to those of the small sweeteners [10]. However, a fundamental problem with all existing models is that it is difficult to explain why proteins are several orders of magnitude sweeter than small molecular mass compounds [13]. At the same time, this aspect makes structure–activity studies of sweet proteins very promising for the design of entirely new sweeteners, possibly mimicking parts of the sweet proteins, if we were able to find a structural entity hosting all essential glucophores, i.e. the mentioned sweet finger. In spite of several attempts, the glucophores of sweet proteins have not yet been identified with certainty, even for brazzein, monellin and thaumatin, the only sweet proteins of known structure [14,15]. Consistently with the idea of a sweet finger, most of these structure-activity studies

Correspondence to P. A. Temussi, Dipartmento di Chimica, 1 Universita` di Napoli, via Cinthia, 80126 Napoli, Italy. Fax: +3 908 167 4409, Tel.: +3 908 167 4416, E-mail: [email protected] Abbreviations: MNEI, monellin; TFE, trifluoroethanol. (Received 23 February 2004, accepted 7 April 2004)

Keywords: models; NMR; sweet proteins; sweeteners; taste receptors.

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searched for a few critical residues, mainly by means of mutagenesis of single or multiple residues [14,16–19]. We have addressed the problem from a very general point of view by means of a surface survey of MNEI, a single chain construct of monellin, with a paramagnetic probe [15], and by studying G16A, a structural mutant of MNEI in which an apparently minor structural change inside the hydrophobic core causes a huge decrease of biological activity [20]. Both studies, while not disproving the hypothesis of a sweet finger, hint that the glucophores might be spread on a surface larger than that of a sweet finger. However, there is no comparative study addressing the possible existence on the three proteins of similar structural features compatible with a sweet finger. The same ELISA study identified that loop L34 of monellin pointed to residues Tyr57-Asp59 of thaumatin at the tip of a possible sweet finger [21]. No similar feature was ever identified for brazzein, where potential glucophores seem to be dispersed over the whole protein surface [19]. Here we present an extensive search for ‘sweet fingers’ on the surface of monellin, thaumatin and brazzein, the three sweet proteins best characterized from a structural point of view. As a result of the search three potential ‘sweet finger’ peptides were designed, synthesized and studied from a conformational point of view.

Materials and methods

Waters PrepLC 40 mm Assembly column C18 (30 · 4 cm, 300 A, 15 mm spherical particle size column). The column was perfused at a flow rate of 40 mLÆmin)1 with a mobile phase containing solvent A (water in 0.1% trifluoroacetic acid), and a linear gradient from 5 to 70% of solvent B (acetonitrile in 0.1% trifluoroacetic acid) in 25 min was adopted for the elution of the peptides. The pure fraction was collected to yield a white powder after lyophilization. Analytical HPLC analyses were performed on a Beckman 125 liquid chromatography fitted with a Alltech column C18 (4.6 · 150 mm 5 mm particle size) and equipped with a Beckman 168 diode array detector; using the above solvent system (solvents A and B) programmed at a flow rates of 1 mLmin)1 with a linear gradient from 0 to 50% or 0 to 80% B in 25 min. Molecular mass of the compounds were determined by a MALDI-TOF analysis using a Hewlett Packard G2025A LD-TOF system mass spectrometer and a-cyano-4-hydroxycinnamic acid as a matrix. For the synthesis of the cyclic derivatives, the linear purified analogue was dissolved in a mixture of H2O/ dimethylsulfoxide/trifluoroacetic acid (75 : 25 : 0.1, v/v) at the concentration of 1 mgÆmL)1. Usually, the cyclization reaction proceeded completely within a day and was monitored by analytical HPLC and Ellman test [25]. After partial evaporation of the solvent, the product was purified by preparative HPLC as mentioned above to yield the desired cyclic compound.

Peptides c[CYFDDSGSGIC(56–66)] (T_YI), c[CLYVYASDKL FRAC(61–73)] (M_LA) and c[CFYDEKRNLQC(37–47)] (B_FQ) were synthesized according to published methods using standard solid-phase synthesis techniques [22] with a Milligen 9050 synthesizer. Protected amino acids and chemicals were purchased from Bachem, Novabiochem or Fluka (Switzerland). The resin loaded with cysteine on the polyethylene glycol/polystyrene support (Fmoc-Xaa-PEG-PS; Xaa ¼ Cys(Trt), Lys(Boc), Glu(OBut) was from Millipore (Waltham, MA, USA). Na-Fmoc derivatives of amino acids were used in the coupling reactions and all lateral amino acid protections were trifluoroacetic acid labile. Fmoc-Xaa-PEG-PS resin (0.5 g in all synthesis) was treated with piperidine (20%) in dimethylformamide and the Na-Fmoc amino acid derivatives (four-fold excess) were sequentially coupled to the growing peptide chain by using [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate] [23] (four-fold excess) in dimethylformamide, and the coupling reaction time was 1 h. Double coupling was required in the acylation step of Ile and Leu. Piperidine (20%) in dimethylformamide was used to remove the Fmoc group at all steps. After deprotection of the last Na-Fmoc group, the peptide resin was washed with methanol and dried in vacuo to yield the protected peptidePEG-PS-Resin. All the protected peptides were cleaved from the resin by treatment with trifluoroacetic acid/H2O/ phenol/ethanedithiol/thioanisole (reagent K) (82.5 : 5: 5: 2.5 : 5; v/v) 10 mL per 0.5 g of resin at room temperature for 1 h [24]. After filtration of the exhausted resin, the solvent was concentrated in vacuo and the residue triturated with ether. Crude peptides were purified by preparative reversedphase HPLC using a Water Delta Prep 4000 system with a

NMR The samples for NMR analysis were prepared by dissolving the appropriate amount of peptide in 18.8 mM KH2PO4 90 : 10 v/v H2O/D2O solution, up to a final concentration of 2 mM. pH was adjusted either to 2.9 or to 6.6. The peptides have also been analyzed in a mixed solvent, adding to the above solution trifluoroethanol (TFE)-2,2-d2 (Aldrich) up to a 37 : 63 (v/v) alcohol/water ratio. NMR measurements were performed on a Bruker DRX500 spectrometer at a temperature of 300 K. Data processing was performed with NMRPIPE [26] and spectral analysis with NMRVIEW [27]. The 1H chemical shifts are relative to trimethylsilylpropionic 2,2,3,3-d4 acid sodium salt as 0.0 p.p.m. Water suppression in the NMR spectra was achieved either by presaturation or by using the WATERGATE pulse sequence [28]. One-dimensional spectra were acquired using typically 32–48 scans with 32 K data size. Data size of two-dimensional spectra was typically 512 · 2048 complex points in the t1 and t2 time-domains, which was zero-filled to obtain 2048 · 4096 data points in the spectrum. Time 2 proportional phase incrementation was applied to achieve quadrature detection in the virtual dimension [29]. Resonance assignments were obtained from two-dimensional experiments. All spin systems were identified by means of total correlation spectroscopy (TOCSY) experiments and all resonances were sequentially assigned by the combined use of TOCSY and nuclear Overhauser enhancement spectroscopy (NOESY). TOCSY spectra were collected with mixing times in the range 50–75 ms, using the DIPSI 2-rc mixing scheme [30]. The NOESY spectra [31] were recorded with mixing times of 200, 300 and 400 ms.

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Model building Model building was performed by means of the program 3 CHEM3D (CambridgeSoft, Cambridge, MA, USA). The starting model of c[CFYDEKRNLQC(37–47)] was derived from the coordinates of the corresponding linear peptide of the parent protein (2brz.pdb). Cycle closure was optimized interactively by monitoring the S–S distance between the side chains of C37 and C47 as a result of slight modifications of all /- and w-values. The resulting cyclic peptide was energy minimized using a simple MM2 force field in vacuo with a dielectric constant of 15, and /start values as the only restraints (Table 1). Unrestrained energy minimization led to a final conformation consistent with that of the corresponding protein loop. Sensory analysis Appropriate amounts of all peptides were dissolved in distilled H2O to make concentrations similar to 10% (w/w) sucrose. The solutions were adjusted to pH 6.6 with 0.1 M NaOH or HCl. The taste panel consisted of four females and three males (age 20–64 years), nonsmokers of reported good health and normal sense of taste. The subjects tasted three peptides, monellin (MNEI) and G16A as positive controls, and deionized water as the negative control in double blind experiments. The sample volume was 150 lL. The solutions were delivered with a micropipette to the anterior part of the subject’s tongue. The subject tasted the compound without any time constrains, then spat it and rinsed with tap water within a 1-min interval. Each subject tested each stimulus three times, and the presentations were randomized. Between each presentation the subjects were asked to score the sweetness of the stimulus on an arbitrary scale. The scale is comprised between ‘barely detectable’, ‘weak’, ‘moderate’, ‘strong’ and ‘very strong’. Experiments were carried out with 4 the understanding and written consent of all subjects.

Results ‘Sweet fingers’ design It is customary to try to identify corresponding parts of proteins with the same function by comparing their sequences or their three-dimensional structures. We have Table 1. Dihedral angles of peptide B_FQ (/nmr) corresponding to sequence CFYDEKRNLQC(37–47) of brazzein (/prot), starting model (/start) and final model (/min). Dihedral angles are calculated according to Ludvigsen et al. [45]. Residue

/nmr

/prot

/start

/min

Phe Tyr Asp Glu Lys Arg Asn Leu Gln

)91 )91 30 )102 )120 )138/60 )138 60 )138

)96 )99 50 )64 )105 66 )139 65 )143

)90 )90 30 )90 )90 60 )90 60 )90

)86 )81 40 )107 )109 77 )91 55 )94

compared the sequences of all known sweet proteins and analyzed the structures of brazzein, monellin and thaumatin. No sequence homology could be detected among miraculin [32], monellin [33], thaumatin [34], curculin [35], mabinlin [36] and brazzein [37]. When a multiple alignment of these sequences was performed by CLUSTAL X [38], the percentages of identical residues, between monellin and the other proteins, measured by the PHYLIP distance matrix was comprised of 23% between monellin and miraculin and a bare 7% between monellin and curculin. There is little similarity among the tertiary folds of the three proteins: when we performed a three-dimensional search of each of the three known structures (brazzein, monellin and thaumatin) against the whole database by means of DALI, an algorithm for optimal pairwise alignment of protein 5 structures [39], the other two proteins were not found. The only elements common among the three proteins are single secondary traits, notably short b-sheet loops. Thus, the essential features of protein substructures that make them potential sweet fingers are: (a) protruding structural features of sufficient length; (b) structural elements having similar counterparts in the other two proteins; and (c) sequences hosting residues consistent with glucophores already identified in small sweeteners. What is the minimum length of a candidate substructure? The models of the receptor [2,40] are inevitably low resolution models as the only available template is an X-ray structure at 2.2 A˚ resolution and a low sequence homology with T1Rs; however, even at low resolution it is relatively easy to identify the location of the active site of the receptor if not the fine details inside it; thus, if one looks at the T1R3 protomer of the heterodimeric model [40] it is clear that the active site, corresponding to the glutamate site of the template, is located at the bottom of a deep cleft, 20–30 A˚ from the surface of the protein. Likely candidates for protruding elements can be searched among substructures with a sufficiently stable secondary structure (otherwise they would be indistinguishable from linear peptides and amino acids, with a low sweetening power); thus, we can restrict our search to loops connecting b-strands or to isolated helices). If we do so, it is immediately clear that there are not many alternatives for consistent choices of loops or other small structural elements common to all three proteins. Figure 1 shows the ribbon representations of the three sweet proteins of known structure. Brazzein represents the simplest case for the choice of a candidate sweet finger as its structure is very simple, with but one loop (Fig. 1A). If one takes into account also side chains, the length of loop L23 is  20 A˚, consistent with the steric requirements outlined above. Strictly speaking, in the case of MNEI (Fig. 1B), a single chain monellin, there is no suitable candidate. Loop L23, centered around Gly51-Phe52, can be excluded immediately as it is not present in native monellin: the dipeptide was added to the sequence of the construct to make a single chain monellin. Loop L45, is an integral part of a rigid b-sheet and accordingly lacks the necessary mobility to act as a flexible finger probing the interior of the receptor. Its involvement as a sweet finger would imply the disruption of the protein’s architecture, with a major energy expenditure. The only remaining candidate, loop L34, is not completely free, but it is structurally similar to the L23 loop of brazzein

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Fig. 1. Comparison of potential sweet fingers of sweet proteins. (A) Loop L23 of brazzein. (B) Loops L23, L34 and L45 of monellin. (C) Loops L23, L34, L45, L67 and L89 of thaumatin. The models were generated by MOLMOL [47].

and, in addition, is the original sweet finger proposed by Kim et al. [21]. Accordingly we chose this loop to design a cyclic peptide for monellin. In the case of thaumatin there are numerous loops with sufficient length to probe the receptor’s cavity. The main ones are labeled in Fig. 1C. However, once again most of them are tightly bound to the body of b-sheet that forms the architecture of this protein and thus, cannot be freed for probing the receptor interior without disrupting the structure of the protein. The only loop that is not tightly bound to the body of b-sheet is loop L67. It is very interesting that this loop is also the one identified by DALI as the only

structural element similar to corresponding ones in brazzein (L23) and in monellin (L34). As a final check, in order to ascertain whether the sequences of the three loops host residues consistent with glucophores already identified in small sweeteners, we can make use of the fact that all models of the active site of the receptor predict the presence, in the finger, of two groups involved in hydrogen bonding with the AH-B entity of the receptor, and of an apolar group at a precise spatial location with respect to the hydrogen bonding groups [10]. According to our indirect model of the active site of the receptor, illustrated schematically by the cartoon of Fig. 2A [10], the relative orientation of an aromatic ring (e.g. belonging to either a Tyr or a Phe in peptides and proteins) should be in a relative spatial orientation, with respect to a pair of hydrogen bond donors or acceptors, similar to that found in aspartame. For instance, in aspartame the aromatic ring is that of Phe, the acceptor is furnished by the side chain of Asp and the donor is the amino group of Asp. It is fairly easy to identify similar groups among the residues of the proposed loops. Figure 2 shows the comparison of the model of aspartame, inserted in the outline of the proposed model (A) of the active site of the receptor [10] with those of potential ‘sweet fingers’ of brazzein (B), monellin (C) and thaumatin (D). In comparing the models, it is clear that in the proteins’ loops the three elements (aromatic ring, acceptor and donor) are not already in a relative spatial position identical to that of the corresponding elements in aspartame, but it is fair to say that the proteins’ models reproduce a static situation whereas generous allowance should be made for side chain rearrangements inside the receptor. Accordingly, it is not difficult, at modest energy costs, to put hydrogen bond donors/acceptors of the three fingers at the same distance of the carboxyl and amino groups of Asp and the aromatic ring of Phe in aspartame. The similarity of the spatial arrangement of the three main glucophores of aspartame and that of the side chains of residues Y65, D68 and K69 of MNEI had been noted even before the complete solution structure of MNEI had been solved [41]. The loop of thaumatin chosen as a potential sweet finger (C56-C66) contains already an S–S bridge joining the two Cys in the protein structure, a circumstance that hints that the corresponding cyclic peptide c[CYFDDSGSGIC(56–66)] might naturally retain the main structural features of the protein loop. Similarly, the loop of brazzein contains two spatially close Cys residues, albeit not connected directly (C37-C47). The loop of monellin does not contain Cys residues, but the residues at the two ends (Q61 and D73 in MNEI) are close enough in space to allow the formation of an S–S bridge if substituted with Cys. Starting from the sequences of the loops we have synthesized three peptides corresponding to the sequences of the loops: c[CYFDDSGSGIC(56–66)] (dubbed T_YI, for thaumatin), c[CLYVYASDKLFRAC(61–73)] (M_LA, for MNEI) and c[CFYDEKRNLQC(37–47)] (B_FQ, for brazzein. In all cases, cyclization is assured by an S–S bridge. Conformational analysis We examined their constitution and their conformational state in solution, mainly to check whether cyclization might

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Fig. 2. Comparison of the model of aspartame with those of potential ‘sweet fingers’ of brazzein, monellin and thaumatin. (A) Ball and stick molecular model of aspartame, inserted in the outline of the old model of the active site of the receptor based on rigid low-molecular mass sweeteners [10]. The atoms of the Phe ring are colored cyan, the acceptor atoms of Asp are colored red, and the donor atom of Asp is colored blue. The shape of the active site is indicated by a simple line. The AH and B groups of the receptor are shown as a blue ball and a red ball, respectively. (B) Neon molecular model of loop CFYDEKRNLQC(37–47) of brazzein. The backbone of the loop is shown in gold, the aromatic side chain of Tyr39 is in cyan, that of E41 in red and that of R43 in blue. (C) Neon molecular model of loop QLYVYASDKLFRAD(61–73) of MNEI. The backbone of the loop is shown in gold, the aromatic side chain of Tyr65 is in cyan, that of D68 in red and that of K69 in blue. (D) Neon molecular model of loop CYFDDSGSGIC(56–66) of thaumatin. The backbone of the loop is shown in gold, the aromatic side chain of Phe58 is in cyan, that of Asp60 in red and that of Ser61 in blue. The models of the loops were generated by MOLMOL [47].

have induced a rigid conformation incompatible with the relative orientation of the three essential glucophores of each loop. All peptides were studied in solution conditions similar to those employed in the original study of MNEI, i.e. in 18.8 mM potassium phosphate at pH 2.9 and at 300 K. NMR experiments were repeated also at pH 6.6, corresponding to the conditions of the sensory analysis. No significant difference with respect to the spectra at pH 2.9 was detected. As the stability of secondary structure elements can be greatly enhanced by solution conditions, the spectra were also run in TFE/water (37 : 63, v/v) solutions at 300 K. Aqueous solutions of TFE are commonly employed in conformational studies of peptides to stabilize helical conformations but it has been shown that they can also greatly stabilize isolated b-sheet loops when these structures are naturally favored by proper residues in the sequence [42]. Assignment of proton resonances was performed in the conventional manner by the standard protocol based on the use of DQF-COSY, TOCSY and NOESY spectra [43]. Apart from obvious chemical shift changes, the spectra in TFE/water do not show diagnostic differences with respect to those in potassium phosphate buffer. NOESY spectra do not hint a single, well defined, ordered tertiary structure for any of the peptides examined. There are however, indications of nascent b-sheet for all three peptides. The three peptides corresponding to hairpin loops of the parent proteins have NMR parameters

consistent with b-strands, even if the stability of the isolated loop is not sufficient to allow the build up of all diagnostic NMR parameters. As shown by Fig. 3, the NH chemical shifts are spread over substantial ranges and the values of JNHCH coupling constants are not centered around 6–7 Hz, as is the case for random coil peptides. In particular, peptide B_FQ derived from brazzein, whose spectrum is shown in Fig. 3C, is characterized by the following JNHCH values (in Hz): 7.8 for F38 and Y39, 5.4 for D40, 8.8 for E41, 9.8 for K42, 8.8 for R43 and N44, 7.3 for L45 and 8.8 for Q46. These values still reflect an average between a few structures as they are not accompanied by a sufficient number of NOEs to allow a straightforward calculation of a single structure. However, they are significantly different from typical random coil averages [44] to hint at the possibility of identifying a representative nascent conformation. We built a model based on the JNHCH coupling constants and compared it with the corresponding loop of the parent protein. In order to build a good starting model, we adopted / angles close to those that can be back calculated from experimental JNHCH coupling constants using a Karplus equation suggested by Ludvigsen et al. [45]. Table 1 shows a comparison of / angles of the final model (/min) with those of the loop in the parent protein (/prot), those derived from NMR (/nmr) and those used in the starting model before energy minimization

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Fig. 3. Partial 1D 1H NMR spectra of the three peptides. (A) NH region of the 1D 1H NMR spectrum of M_LA {c[CLYVYASDKL FRAC(61–73)]}. (B) NH region of the 1D 1H NMR spectrum of T_YI {c[CYFDDSGSGIC(56–66)]} (C) NH region of the 1D 1H NMR spectrum of B_FQ (c[CFYDEKRNLQC(37–47)]}. All spectra were recorded at 500 MHz at 300 K, using approximately 2 mM solutions in 18.8 mM KH2PO4 90 : 10 (v/v) H2O/D2O at pH 2.9.

Fig. 4. Comparison of the representative solution conformation of peptide B_FQ {c[CFYDEKRNLQC(37–47)]}} with the parent protein structure. (A) Histograms of the JNHCH coupling constants of the final model (blue), the experimental ones (red) and those of the corresponding protein loop (ivory). JNHCH values were calculated according to Ludvigsen et al. [45]. (B) Superposition of the conformations of the final model and of the protein loop using only backbone atoms of residues FYDEKRNLQ(38–46).

cases the NMR data are consistent with the presence, in solution, of mixtures of closely related hairpins, as one would expect in the case of a limited frame-shift along the cycle. Sensory analysis

(/start). After cycle closure with the proper S–S bond between C37 and C47, the cyclic peptide was energy minimized using a simple MM2 force field in vacuo and using /start values as the only restraints. Unrestrained energy minimization led to a final conformation consistent with that of the corresponding protein loop. Figure 4A shows a comparison of the JNHCH coupling constants of the final model and of the protein loop, calculated according to Ludvigsen et al. [45] with experimental ones. Figure 4B shows the superposition of the conformations of the final model and of the protein loop using only backbone atoms of residues FYDEKRNLQ(38–46). It is clear that the cyclic peptide can assume in solution a conformation very similar to that of the corresponding loop in the protein, although the turn is shifted by one residue. It is not possible to build models of similar quality for the other two peptides, but it is safe to state that all three cyclic peptides show a clear tendency to assume hairpin conformations in aqueous solution. In all

All peptides were assayed by a panel of trained tasters who classified their sweet taste according to a scale ranging from ‘barely detectable’ to ‘very strong’. All three peptides were found devoid of any sweet taste, even at high concentrations.

Discussion Sweet fingers We have designed three cyclic peptides corresponding to hypothetical ‘sweet fingers’ of brazzein, monellin and thaumatin. None of these peptides is sweet, even at high concentrations. In principle, the lack of sweet taste can be attributed to a wrong primary structure (i.e. the chosen sequence does not contain all residues necessary to trigger response), to the lack of ordered secondary structure (i.e. to the fact that the restraints imposed by cyclization do not correspond to those present in the three-dimensional

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structure of the parent protein or worse prevent the peptide to attain the native conformation) or a combination of the two (e.g. the sequence contains the essential elements of a sweet finger but the position of the S–S bridge distorts the conformation). The peptides designed in the present study show a clear tendency to assume extended conformations consistent with those present in the parent proteins. The residual flexibility allows the attainment of closely related hairpin conformations. That is, the peptides explore a limited portion of conformational space that includes the right native conformation. It is conceivable that they could interact with the receptor via an induced-fit mechanism. None of the cyclic peptides designed to mimic ‘sweet fingers’ is able to elicit sweet taste, and as there are no obvious alternative choices for putative ‘sweet fingers’ in the proteins, it is necessary to look for alternative explanations of the high biological activity of the parent proteins. The presence of ‘sweet fingers’ is not the only possible explanation of the sweet taste of proteins. On the basis of docking calculations to a model of the T1R2-T1R3 receptor, we have recently proposed that all sweet proteins of known structure, i.e. brazzein, monellin and thaumatin, interact the T1R2-T1R3 receptor in a way completely different from that of small molecular mass sweeteners [40]. The wedge model The sweet taste receptor has been classified as a metabotropic transmembrane receptor with a marked similarity to the dimeric metabotropic glutamate m1-LBR receptor [2]. The similarity between the sequences of the two chains of the T1R2-T1R3 receptor and that of the single chain of the homodimer of the m1-LBR mGlu receptor is sufficient to allow model building [40] and to assume that it has the same general features. The structure of the extracellular N-terminal domain of the m1-LBR mGlu receptor was solved by X-ray diffraction and shown to exist in three different crystal forms [46], one complexed with the ligand (1ewk.pdb) and two, free form I (1ewt.pdb) and free form II (1ewv.pdb), without ligand. Free form I, an ‘inactive’ conformation rather different from the conformation of the complex, is in equilibrium with free form II whose conformation is nearly identical to that of the ‘active’, complexed form. If the T1R2-T1R3 receptor behaves like the m1-LBR mGlu receptor, it should also exist as a mixture of three forms: a complexed form containing a low molecular mass sweetener (corresponding to a molecule of glutamate), free form I, the ‘inactive’ conformation and free form II, whose structure is nearly identical to that of the ‘active’, complexed form. As shown in Fig. 5A, the ‘normal’ way to activate the receptor is by the binding of a small molecular mass sweetener, e.g. aspartame, that transforms free form I into the complexed form. However, the equilibrium between form I and form II can also be shifted in favor of form II if we can stabilize it in another way. Figure 5B illustrates how stabilization can be achieved by external binding of a macromolecule on a secondary binding site on the surface of the receptor. The actual feasibility of this binding was checked by docking calculations of brazzein, monellin and thaumatin to a model receptor that can be built using the structure of form II of

m1-LBR as a template [40]. It was shown that all three sweet proteins fit a large cavity of the receptor with wedge-shaped surfaces of their structures. Efficient binding is assured both by shape and charge complementarity as the cavity is predominantly negative and the interacting surfaces of the proteins are mainly positive. The first experimental evidence, albeit indirect, in favor of this mechanism came from the solution structure of G16A, a structural mutant of MNEI in which an apparently minor structural change inside the hydrophobic core causes a tenfold decrease of biological activity [20]. The fact that the cyclic peptides described in the present, possessing all requirements to act as small molecular sweeteners, i.e. favorable conformational properties and the presence of the right glucophores, are not able to interact with the receptor, can be taken as further evidence in favor of the alternative mechanism [40]. The main reason of their inability to occupy the small cavity corresponding to the glutamatehosting site of the m1-LBR receptor is probably the accessibility of this site. In fact, in the case of the m1-LBR receptor the two cavities that bind glutamate are rather buried [46]. This is the major difference with respect to the model active site we proposed on the basis of rigid molecular molds [10], because, in that model, a necessary requirement for interaction with ‘sweet fingers’ was unhindered accessibility. It might be argued that the cyclic peptides could interact with the secondary binding site [40] as the intact parent proteins. In order to interact with the secondary site as the intact protein, each peptide should be able to cover a substantial portion of the interactive surface. Therefore, it

Fig. 5. Cartoon illustrating the two possible mechanisms of interaction of sweet molecules with the sweet taste receptor. (A) The binding of aspartame transforms inactive, ligand-free form I into the complexed form of the receptor, which activates signal transmission. (B) A sweet protein binding to a surface site of the ligand-free form II shifts the equilibrium between inactive free form I and active free form II in favor of the latter. Form II, stabilized by protein complexation, activates long lasting signal transmission.

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Fig. 6. Models of representative complexes of the proteins with free form II of the model receptor T1R2-T1R3. The residues of the sweet proteins and of the receptor model are represented by atoms: The proteins’ atoms are colored green, except for residues corresponding to potential ‘sweet fingers’ that are colored gold. Those of the T1R2 protomer are colored pale pink, those of the T1R3 protomer dark pink. (A) The left-hand side shows a complex of brazzein with free form II of T1R2-T1R3. The right-hand side shows a view of the same complex rotated by 180 around a vertical axis. The model of T1R2-T1R3 in the right-hand side panel is a line model to expose the inner side of interacting surface of brazzein. (B) The left-hand side shows a complex of MNEI with free form II of T1R2-T1R3. The right-hand-side shows a view of the same complex rotated by 180 around a vertical axis. The model of T1R2-T1R3 in the right-hand-side panel is a line model to expose the inner side of interacting surface of MNEI. (C) The left-hand-side shows a complex of thaumatin with free form II of T1R2-T1R3. The right-hand-side shows a view of the same complex rotated by 180 around a vertical axis. The model of T1R2-T1R3 in the right-hand-side panel is a line model to expose the inner side of interacting surface of thaumatin. All models were generated by MOLMOL [47].

may be interesting to see their relative positions on the surface of interaction of the parent protein. Figure 6 shows representative complexes of form II of the T1R2-T1R3 model with the structures of the three sweet proteins. The left-hand side panel of Fig. 6A shows the outer face of a complex of brazzein. A part of the CFYDEKRNLQC(37– 47) loop, colored gold, is in contact with the medium, i.e. it is not interacting with the receptor. The right-hand side panel shows a view of the brazzein complex, rotated by 180 around a vertical axis, in which the receptor model is shown with only a line representation of the backbone to expose the surface of brazzein that is in contact with the receptor. It can be seen that the area of the portion of loop CFYDEKRNLQC(37–47) interacting with the receptor represents a small fraction of the total interacting surface. The left-hand-side panel of Fig. 6B shows the outer face of a complex of MNEI. The QLYVYASDKLFRAD(61–73) loop is completely hidden from the outer environment. The right-hand-side panel shows a view of the MNEI complex,

rotated by 180 around a vertical axis, in which the receptor model is shown with only a line representation of the backbone to expose the surface of MNEI that is in contact with the receptor. It can be seen that loop QLYVYASDKLFRAD(61–73) is partially buried inside the structure of MNEI leading to a fairly small interacting area with respect to the total interacting surface. The situation depicted in Fig. 6C for thaumatin is similar to that of MNEI. Also in this case, the left-hand side panel of Fig. 6C shows that the CYFDDSGSGIC(56–66) loop is completely hidden from the outer environment, whereas the visible portion in the right-hand-side panel is even smaller, leading to a negligible interacting area with respect to the total interacting surface. In summary, we can conclude that the data presented in this paper do not support the idea of a localized ‘sweet finger’ site that could explain the properties of sweet proteins according to the rules established for small sweet molecules. Alternative explanations that could better explain the

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experimental observations, such as the ‘wedge model’ recently proposed by us [40], should therefore be explored. 21.

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