Chapter 36
How Sweet It Is: Detailed Molecular and Functional Studies of Brazzein, a Sweet Protein and Its Analogs 1,2
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Fariba Assadi-Porter , Marco Tonelli , James T. Radek , Claudia C. Cornilescu , and John L. Markley
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Department of Biochemistry, National Resonance Facility at Madison, Department of Entomology, and Center for Eukaryotics and Structural Genomics, University of Wisconsin-Madison, Madison, WI 53706 *Corresponding author:
[email protected] 3
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Brazzein is a small, low-calorie, sweet protein with high stability over wide temperature and p H ranges. Brazzein has desirable taste characteristics that resemble those of carbohydrate sweeteners. Brazzein folds in a β-α-β topology in which the α-helix packs against the three-stranded antiparallel β-sheet. This structure is held together by four disulfide bridges. We developed an efficient bacterial production system for brazzein that allows us to express wild -type and mutant proteins. We have designed a large number of brazzein variants for taste tests. These include mutations that affect surface charges, disulfide bridges, loops, and flexible regions. We have subjected a subset of these variants to detailed analysis by N M R spectroscopy to identify patterns of hydrogen bonds and internal mobility. The results show a correlation between these physical properties and the sweetness of the protein. These results led us to propose a multi-site binding model for the interaction between brazzein and the heterodimeric human sweet receptor, which we are continuing to test with the goal o f designing more potent brazzein analogs as potential future sweeteners. 2
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© 2008 American Chemical Society
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Historical background The discovery of a number of non-calorigenic sweet proteins over the last 30 years has increased the demand for non-carbohydrate-based sweeteners with favorable taste properties. The optimal design of such sweeteners requires knowledge about structure-function relationships and the identification of chemical entities that trigger the sweetness response. Proteins with sweetness ranging from 100 to 3000 times that of sucrose on a weight basis have been identified in African and South Asian fruits and berries. Among the known, naturally occurring, sweet-tasting proteins, brazzein has properties that make it particularly attractive as a potential economic sweetener. Brazzein comes from a West African plant Pentadiplandra brazzeana, a climbing vine that grows in Gabon, Zaire, and Cameroon (/). The pulp of the ripe berries contains brazzein at about 0.2 % by weight, making the berries intensely sweet. The fruit, locally named "j'oublie", is consumed by the local population and is prized for its sweetness. Brazzein is highly stable over wide temperature and pH ranges and has taste properties that resemble those of carbohydrate sweeteners. The brazzein protein is a single polypeptide chain composed of 54 standard amino acids and contains no carbohydrate. Brazzein has been shown to elicit sweetness responses in humans by taste trials and in a non-human primate (rhesus monkey) as determined from electrophysiological recordings of signals from chorda tympani nerve fibers (2).
Human Taste Receptors The human receptor appears to be a heterodimer of two conventional seventransmembrane-helix G-coupled type receptors (T1R2/T1R3), each with an unusually large ectodomain (3, 4). T1R2+T1R3 receptors are the primary sweet receptors for a diverse range of sweet ligands (3-6). Calcium imaging assays of H E K cells transfected with hTlR2+hTlR3 respond to all sweet taste stimuli tested: sugars sucrose, fructose, galactose, glucose, etc.; amino acids glycine and D-tryptophan; sweet proteins brazzein, monellin and thaumatin; and synthetic sweeteners cyclamate, saccharin, ace K , aspartame, dulcin, neotame and sucralose (3, 6-10). A l l of these responses are inhibited by the sweet taste inhibitor lactisole, which acts on the transmembrane domain (TMD) of T1R3 (8, 10, 11). Different families of ligands interact with different sites on the receptor. It has been shown that TIRs in both umami and sweet taste share a common subunit (T1R3) (10). In addition, Jiang et al. recently reported that the C-rich region of the extracellular domain of the T1R3 subunit of the sweet taste receptor plays an important role in differentiating responses to brazzein (6). Interestingly, human-specific sweet proteins are recognized only by the human T1R2/T1R3 receptors but not the rat T1R2/T1R3 receptors (4), as consistent
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with the previous finding that brazzein does not excite taste fibers in the rat (12). Although tentative models have been proposed for interactions of the sweet receptor with sweet proteins and small sweet ligands (13), the mechanism is not understood. The discovery and characterization of the sweet taste receptor opens up exciting new avenues for research on the detailed mechanism of action of sweet substances. Brazzein is an excellent candidate for experimental investigations of the chemical and structural requirements for extracellular triggering of a sweet response in humans and for understanding the mechanism of the signal transduction.
Early investigations of brazzein isolated from the fruit The amino acid sequence of brazzein was determined by peptide sequencing (/). Brazzein extracted from fruit is composed of at least two species, one fraction containing a pyroglutamyl (pGlu) N-terminus and a second, sweeter, fraction lacking the pGlu residue (14). Early variable temperature N M R studies of brazzein showed very little change in its H N M R spectrum over a wide range of temperature range (32-82 °C) (75),and the sweetness profile was shown to be undiminished after incubation of brazzein at 100 °C for four hours (16). The three-dimensional structure of pGlu-brazzein extracted from fruit was solved by homonuclear H N M R spectroscopy with R M S D = 1.6 Â (Figure 1) (17) . The protein has a highly compact structure consisting one short ûr-helix and three anti-parallel /^-strands held together by four disulfide bridges. No significant sequence or structural similarity was found between brazzein and the two other sweet-tasting proteins of known three-dimensional structure: monellin (18) , and thaumatin (19, 20). These original N M R studies of brazzein determined that the protein adopts a cysteine-stabilized αβ (CSafi fold in which the α-helix and ^-strands are stabilized by the presence of four disulfide bridges) (17). The structure suggests that this evenly distributed web of disulfide bridges stabilizes the protein by limiting the conformational entropy of its unfolded state. !
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Structural relationship between brazzein and other members containing CSafi fold Other CSar/? fold proteins include members of the rapeseed family of serine proteinase inhibitors, scorpion toxins, insect defensins, plant-derived ^thionins and a family of antimicrobial peptides. Apart from the conserved cysteines, little sequence identity was found between members of the different families.
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Figure 1. Backbone ribbon diagram and the surface representation of the brazzein extractedfromfruitwith positions of disulfide bonds are shown as determined by solution-state HNMR spectroscopy (17). (See color insert in this chapter.) !
Brazzein is the only CSa/7 protein known to be sweet, and it has no proteinase inhibitor activity (21).
Production of recombinant brazzein, stable isotope labeled brazzein, and brazzein mutants The protein shows great promise as a natural low-calorie sweetener. To understand the structural and chemical properties responsible for its sweetness, we have engineered a synthetic gene to express the brazzein molecule and developed the first production system for brazzein in bacteria (22). We have used this approach to discover mutants with sweet-taste properties that appear to be different than the wild-type protein (23). The sequence of the recombinant protein product is identical to the minor form of brazzein isolated from fruit, the form that lacks the N-terminal pyro-glutamate (pGlu) residue (we refer to this product, des-pGlul -brazzein, henceforth as wild-type brazzein). Recombinant wild-type brazzein has about twice the sweetness of brazzein extracted from the fruit, which is primarily the pGlu variant.
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564 We make the protein as a fusion with a modified form of the enzyme staphylococcal nuclease. The fusion protein is expressed in Escherichia coli as an insoluble product, which we solubilize, fold, and cleave to yield des-pGlulbrazzein. The conditions we developed for folding and oxidation of the disulfides lead to a product with native structure (as determined by N M R spectroscopy) and with full activity as a sweetener (as determined by taste tests) (22). Production o f brazzein from E. coli has also enabled us to make samples labeled with stable isotopes ( N or C / N ) for N M R investigations of the structure and dynamics of the protein. We recently solved a higher resolution N M R structure of wild-type brazzein by more modern methods by using C / N double labeling with R M S D = 0.38 A (C. Cornilescu and F. M . Assadi-Porter, in preparation). The C/ N-double labeled brazzein was produced by our nuclease expression system. The stable isotope labeling enabled us to determine structural restraints from long-range NOEs and residual dipolar couplings required for high resolution N M R structure determination. The structure obtained from recombinant brazzein shows the same secondary structure as the fruit brazzein but with more refined backbone and side chain conformations. 1 5
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Summary of results from mutagenesis of brazzein Our first mutagenesis design was based on alanine scanning of solvent exposed residues. Our results from the variants, prepared, purified and subjected to threshold analysis by taste tests (Figure 2) (22, 24, 25) revealed the presence of multiple critical areas or "interaction sites": regions near Arg43 (Loop43) and the N - and C-terminal domains. Several charged residues in these regions were found to be essential to sweetness, as suggested by previous chemical modification studies (16). Our studies have indicated that the presence of positive charges on the surface of brazzein enhances sweetness: mutating some of these positive charges to neutral or negative charge significantly decreases the sweetness (24, 25). Charged residues also have been reported to be critical for the sweetness of thaumatin and monellin (26-29).
Multisite
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Terminal regions. Both the N - and the C-terminal regions are important for sweetness in brazzein. Insertion of alanine at the N-terminus following the removal of methionine (Alal-Asp2-sequence) increased sweetness and suggests that the presence of a hydrophobic side chain at this position enhances binding to the receptor ((22), and F. M . Assadi-Porter, unpublished results). A t the C-terminus, deletion or addition of any residue
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at position 54 diminished sweetness (deletion of Tyr54 or insertions of arginine residues at positions 55 and 56). Mutations near the C-terminus greatly altered sweetness (Asp50Ala, Cys52Ala) or changed protein stability (for example, TyrSlAla did not fold, probably because of a clash in hydrophobic side chain packing) (Figure 2) (22). Charged residues and loop regions. Replacement of any of the charged residues near the loop regions greatly affected sweetness. Mutants Asp29Ala (Asp29Asn or Asp29Arg), His31A, Glu41Lys increased sweetness two- to four-fold. However, replacement of either Arg33 or Arg43 (at either end of >S-strand III) by Ala decreased sweetness (3-4-fold for Arg33Ala but >50-fold for Arg43Ala) (Figure 2). Asp50Ala (or Asp50Asn) in the /?-strand II near the C-terminus also decreased sweetness 2-3-fold. Mutants Arg33Ala and Asp50Ala, which introduce changes near the C-terminus, caused decreased sweetness. Arg33 interacts with residues near the C-terminus (including Asp50, Tyr51 hydrophobic packing) and near the N-terminus (including Lys5 and Lys6). As described below, these residues participate in backbone hydrogen bonds that may serve to transmit conformational effects of the mutations to adjacent parts of the structure (23, 30, 31).
NMR studies For our detailed structural and dynamic analyses of brazzein variants, we chose wild-type brazzein and five mutants (two with increased sweetness and three with decreased sweetness). The ribbon-diagram in Figure 2 shows the backbone of wild-type brazzein and the positions of the five mutations (31). Four of the sites of mutation (Ala2 insertion, His31Ala, Arg33Ala, and AspSOAla) are spatially close to one another. Two of the mutants (Ala2 insertion and His31Ala) have about twice the sweetness of wild-type brazzein; the other three mutants (Arg33Ala, Arg43Ala, and Asp50Ala) have greatly reduced sweetness (25). Arg43 Ala is essentially tasteless. Only one of the five mutants (Arg43 Ala) exhibited chemical shift changes in regions remote from the site of mutation. In. this mutant, changes in chemical shifts were observed in the N - and C-terminal regions, flexible 9-19 loop, as well as Arg33 loop region.
Hydrogen bonding Thanks to a recent discovery, hydrogen bonds can now be detected and quantified through measurements of N M R spin-spin couplings. HNCO-type
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Figure 2. Surface representation of wild-type brazzein showing a summary of key mutations that change sweetness. Mutations that abolished sweetness are shown in dark blue, whereas those that enhance sweetness are shown in gray. Mutations that slightly intermediate decreased sweetness are shown in lighter blue. Note that the side chains proposed constitute the primary sweet sites (Loop43 and N- and C-terminal regions) are on the same face of the molecule (22, 24, 25). (See color insert in this chapter.)
In Sweetness and Sweeteners; Weerasinghe, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.
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N M R experiments provide information about trans-H-bond J couplings, which have magnitudes usually less than 1 H z (32-34). The magnitudes of the coupling constants can be converted into Η-bond distances. The high sensitivity afforded by a triple-resonance cryogenic N M R probes has improved the practicality of using trans-hydrogen-bond couplings as a screen for monitoring structural changes in a protein backbone (i.e. ''tight" vs. "relaxed" conformations) that result from changes in solution conditions or mutations (35, 36). Such measurements can be carried out much more quickly than a full structure determination typically with resolution > 0.1 Â. Changes in V c couplings identify hydrogen bonds that are strengthened or weakened; missing peaks identify hydrogen bonds that may be broken; and new peaks indicate the presence of new hydrogen bonds will allow us to monitor subtle structural changes. We have used this approach to investigate wild-type brazzein and a series of brazzein variants with altered sweetness properties (30, 31). Application of this approach to wild-type brazzein revealed 17 hydrogen bonds (Figure 3), 13 of the Η-bonds were identical to those deduced from the N M R structure of fruit brazzein (37). Two additional Η-bonds were detected, and two were found to be different from those reported earlier. Hydrogen bond patterns in brazzein indicate one α-helical stretch from residues 20-29 and three antiparallel ^-strands, composed of residues 4-7, 34-40, and 44-51 all consistent with the calculated three-dimensional structure (31). Analysis of the Η-bonds in the six brazzein variants through measurements of trans-H-bond couplings has shown that single-site mutations can give rise to subtle structural changes (31). Interestingly, less sweet variants (Arg33Ala, Arg43Ala, and Asp50Ala) share the loss of two common Η-bonds: Glu36 H —148 O' between /^-strands II and III where they are twisted and Lys27 H —Asn23 O' in the middle of the α-helix. By contrast, our results indicate that the two sweeter forms of brazzein, those with least change in chemical shifts (His31Ala and Ala2 insertion) maintain the wild-type pattern of H-bonds (37). Our Η-bond data indicate that less sweet brazzein variants frequently have stronger Η-bonds between /^-strands than those in wild-type. However, this is not the case with sweeter variants that maintain wild-type like Η-bond patterns. N C
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Dynamics Dynamic analysis by N M R can provide valuable information about the rigidity of proteins and the time dependence of fluctuations in molecular structures. We have carried out a preliminary analysis of N relaxation data collected for brazzein at different N M R field strengths (600 M H z and 900 MHz). As determined by N M R relaxation measurements, mutations that decrease sweetness were found to change the flexibility of the protein (31). In particular, comparison of relaxation parameters from mutant Arg43Ala (non1 5
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Figure 3. Hydrogen-bonds in wild-type brazzein deduced from trans-hydrogenbond-couplings detected by NMR spectroscopy. Wild-type brazzein and two mutants with enhanced sweetness show a common pattern of hydrogen bonds, whereas all three variants with reduced sweetness have common loss of hydrogen-bonding patterns shown in dotted arrow lines (31). (See color insert in this chapter.)
sweet analog) with those from wild-type brazzein indicates that internal mobility is decreased significantly in the loop regions of the mutant protein (Figure 4) (unpublished results). The observed loss of internal mobility can be explained in terms of the earlier trans-H-bond coupling results, which indicated that this mutation leads to a shift in one Η-bond into the loop-43 region and the gain of two Η-bonds between yff-strands I and II (26).
Conclusions Brazzein is a small high potency protein sweetener. In our earlier studies we had proposed a multi-site binding interaction model for brazzein with the hetrodimeric sweet receptors. The mutagenesis studies revealed the presence of at least two sites that are important for the sweetness. These sites are N - and C termini and Loop43 region. Residues found to be important for the sweetness function are located on the same face of the molecule and separated in space by the presence of /2-strands II and III. In general the presence of positive charges or introduction of hydrophobic side chains at the N-terminus enhances sweetness indicating the importance of both electrostatic and Van der Waal requirements for brazzein-sweet receptor interactions.
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Figure 4. Comparison of relaxation parameters (longitudinal Ν relaxation, Tj; transverse Ν relaxation, T ; and Ή- Ν nuclear Overhauser enhancement, NOE) derivedfrom wild-type brazzein (left panel) andArg43Ala (non-sweet mutant) (right panel). The internal mobility is decreased significantly in the loop regions of the mutant protein (F. M. Assadi-Porter, and C.C Cornilescu, unpublished data.) 15
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Our combined investigation of hydrogen bonding and dynamics of wild-type and mutant brazzeins have revealed a correlation between these properties and protein sweetness. These results will be important for interpreting future studies of the interaction between sweet proteins and the sweet taste receptor and for understanding how they are different from interactions of the receptor with small sweet ligands.
Acknowledgments This work was supported by grants RR02301 (JLM) and DC006016 (Goran Hellekant, P.I.).
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