Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Brazzein, a Small, Sweet Protein: Effects of Mutations on its Structure, Dynamics and Functional Properties
National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, 433 Babcock Drive, Madison WI 53706, USA
Correspondence to be sent to: John L. Markley, e-mail: markley{at}nmfam.wisc.edu
Key words: brazzein, conformation, hydrogen bond, mutagenesis, structure–function, sweet taste
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Introduction |
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 Top Introduction Early investigations of brazzeinProduction of recombinant...NMR studiesInvestigation of model peptidesRelationship between brazzein...AcknowledgementsReferences |
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The demand for non-calorigenic protein-based sweeteners with favorable taste properties is high. The optimal design of such sweeteners requires knowledge about structure–function relationships and the identification of chemical entities that trigger the sweetness response. Among the known, naturally occurring, sweet-tasting proteins, brazzein has properties that make it particularly attractive as a potential economic sweetener. It is highly stable over wide temperature and pH ranges and has taste properties that resemble those of carbohydrate sweeteners. Brazzein is a single polypeptide of 54 standard amino acids and contains no carbohydrate. The brazzein protein originally was purified from the fruit of Pentadiplandra brazzeana, a climbing vine that grows in Gabon, Zaire and Cameroon (Ming and Hellekant, 1994
). The fruit is consumed by the
local population and is prized for its sweetness. Pure 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 the chorda tympani
(van der Wel et al.,
1989).
Recently receptor proteins have been discovered that are responsive to sweet ligands.
The human receptor appears to be a heterodimer of two conventional
seven-transmembrane-helix G-coupled type receptors (T1R2/T1R3) but with unusually large
ectodomains ((Nelson et al.,
2001;
Li et al., 2002); P. Jiang
and M. Max, personal communication). Different sweet proteins interact with and activate
the same heterodimeric receptor in somewhat different ways and tentative models for such
interactions have been elaborated ((Temussi,
2002); M. Max, personal communication). The discovery of the sweet taste
heteroreceptor opens up exciting new avenues for research on the 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.
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Early investigations of brazzein |
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TopIntroductionEarly investigations of brazzeinProduction of recombinant...NMR studiesInvestigation of model peptidesRelationship between brazzein...AcknowledgementsReferences |
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The amino acid sequence of brazzein was determined by peptide sequencing (Ming and Hellekant, 1994
) and the
three-dimensional structure of brazzein was solved by homonuclear 1H NMR
spectroscopy (Caldwell et al.,
1998a). The protein has a highly compact structure consisting of one short
-helix and three anti-parallel ß-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
(Somoza et al., 1993) and
thaumatin (Ogata et al.,
1992;
Ko et al., 1994). Early
variable temperature NMR studies of brazzein showed very little change in its
1H NMR spectrum over a wide range of temperatures (32–82°C;
Caldwell et al., 1998b) and
the sweetness profile was shown to be undiminished after incubation at 100°C for 4 h
(Ming et al., 1996).
The original NMR studies of fruit brazzein indicated that the protein adopts a
cysteine-stabilized ß (CSß) fold in which the -helix and
ß-strands are stabilized by the presence of four disulfide bridges (Caldwell et al., 1998a). Other proteins
with this fold include members of the rapeseed family of serine proteinase inhibitors,
scorpion toxins, insect defensins and plant-derived 
-thionins. Apart from the
conserved cysteines, little sequence identity is found between members of the different
families. Brazzein is the only CSß protein known to be sweet.
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Production of recombinant brazzein, stable isotope labeled brazzein and brazzein mutants |
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TopIntroductionEarly investigations of brazzeinProduction of recombinant...NMR studiesInvestigation of model peptidesRelationship between brazzein...AcknowledgementsReferences |
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We developed an efficient bacterial production system for brazzein (Assadi-Porter et al., 2000a
). The
recombinant protein produced has a sequence identical to the minor form of brazzein
isolated from fruit, the form that lacks the N-terminal pyro-glutamate (pGlu) residue
(des-pGlu1-brazzein) and that has been shown to have about twice the sweetness of the
pGlu containing variant. The fusion protein is expressed as an insoluble product, which
we solubilize and fold. The conditions we developed for folding and oxidation of the
disulfides lead to a product with native structure (as determined by NMR spectroscopy)
and with full activity as a sweetener (as determined by taste tests) (Assadi-Porter et al., 2000a).
This methodology, along with quick-change mutagenesis, has allowed us to make a
variety of brazzein mutants. We discovered mutants with sweet-taste properties that
appear to be superior to those of the wild-type protein (Assadi-Porter et al., 2000b). 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 (Jin et
al., 2003a,b).
Production of brazzein from Escherichia coli has also enabled us to make
samples labeled with stable isotopes (15N or 13C and
15N) for NMR investigations of the structure and dynamics of the protein. For
our detailed structural and dynamic analyses of brazzein variants, we chose wild-type
brazzein (des-pGlu1-brazzein) and five mutants (two with increased sweetness and three
with decreased sweetness). The ribbon-diagram in Figure
1 shows the backbone of wild-type
brazzein and the positions of the five mutations (Assadi-Porter et al., 2003). Four of the sites of
mutation (Ala2 insertion, His31Ala, Arg33Ala and Asp50Ala) 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 (Assadi-Porter et
al., 2000b). Arg43Ala is tasteless.
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NMR studies |
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TopIntroductionEarly investigations of brazzeinProduction of recombinant...NMR studiesInvestigation of model peptidesRelationship between brazzein...AcknowledgementsReferences |
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In only one of the five mutants (Arg43Ala) were the chemical shifts changes resulting from the mutation propagated to other parts of the molecule. In this mutant, changes in chemical shifts were observed in the N- and C-terminal regions.
Analysis of the H-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 (Assadi-Porter et al.,
2003). Wild-type brazzein and the two variants with sweetness equal to or
greater than wild-type brazzein had similar patterns of H-bonds, whereas all three
variants with reduced sweetness exhibited changes in H-bonding (Assadi-Porter et al., 2003).
As determined by NMR relaxation measurements, the mutations that decrease sweetness
were found to decrease the flexibility of the protein. The results suggested, in
addition, that loop 9–19 of brazzein exists as two or more sub-structures. We
measured residual dipolar couplings (RDCs) as a means for determining whether loop
regions are disordered. The RDCs from the sweeter brazzein analog (Ala2insertion) were
similar to those from wild-type brazzein; this confirmed that the two proteins have
similar structures. Furthermore, the RDC results indicated that residues 11–18 in
the loop between the first ß-strand and -helix are disordered in both proteins
(F.M. Assadi-Porter and C.C. Cornilescu, unpublished results).
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Investigation of model peptides |
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TopIntroductionEarly investigations of brazzeinProduction of recombinant...NMR studiesInvestigation of model peptidesRelationship between brazzein...AcknowledgementsReferences |
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On the basis of our model for multi-site brazzein:receptor interactions, we designed a small cyclic peptide corresponding to regions of the N- and C- termini connected by a tri-peptide linker (PGN) at one end and a disulfide bond at the other end. The resulting cyclic peptide, c[(D2KCKKV7)-PGN-(D50YCEY54)], was designed to contain a proper ß-turn (type I or II) motif. This conformation was confirmed by homonuclear 1H-1H 2D TOCSY and NOESY NMR data (F.M. Assadi-Porter, unpublished results). In a taste test, however, the peptide was found to be tasteless. Similar results were obtained in the Temussi laboratory on a different cyclic peptide. That study examined the cyclic peptide c[C37FYDEKRNLQC47], which proved to be tasteless (Tancredi et al., 2004
).
Both investigations of model peptides suggest that a more extensive structure is required
for sweetness. |
Relationship between brazzein and proteinase inhibitors |
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TopIntroductionEarly investigations of brazzeinProduction of recombinant...NMR studiesInvestigation of model peptidesRelationship between brazzein...AcknowledgementsReferences |
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The sequence of brazzein is very similar to that of the rapeseed-type proteinase inhibitors from plants and the structures are also similar (Caldwell et al., 1998a
;
Zhao et al., 2002). The
reason why brazzein has no activity as a proteinase inhibitor is explained by its lack of
the reactive site dipeptide (Zhao et
al., 2002). Attempts to convert brazzein into a trypsin inhibitor by
inserting the missing residues or even by introducing the entire reactive site loop of
the Arabidopsis thaliana inhibitor proved unsuccessful; these insertions or
modifications either destabilized the mutated protein or prevented it from folding
(Zhao, 2001; F.M. Assadi-Porter and
J.L. Markley, unpublished data). |
Acknowledgements |
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TopIntroductionEarly investigations of brazzeinProduction of recombinant...NMR studiesInvestigation of model peptidesRelationship between brazzein...AcknowledgementsReferences |
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We thank Dr Marianna Max and P. Jiang for sharing information about their research prior to publication. This work was supported by grants RR02301 (JLM) and DC006016 (Göran Hellekant, P.I.).
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TopIntroductionEarly investigations of brazzeinProduction of recombinant...NMR studiesInvestigation of model peptidesRelationship between brazzein...AcknowledgementsReferences |
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