Proc. Natl. Acad. Sci.
USA
Vol. 82, pp. 1406-1409, March 1985
Biochemistry
Three-dimensional structure of thaumatin I, an intensely
sweet protein
(sweet taste/taste reception/sweet determinants/thaumatin crystal structure)
ABRAHAM M. DE VOS*, MARCOS HATADAt, HENK VAN DER WEL*, H. KRABBENDAM§, A. F. PEERDEMAN§,
AND SUNG-HOU KIMt
tDepartment of Chemistry and Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720; tUnilever Research Laboratory, Vlaardingen,
The Netherlands; and Department of General Chemistry, University of Utrecht, Utrecht, The Netherlands
Communicated by Daniel E. Koshland, Jr., October 31, 1984
ABSTRACT Thaumatin and monellin are the two sweetest
compounds known to man-about 100,000 times sweeter than
sugar on a molar basis and 3000 times on a weight basis. These
proteins represent a unique class of proteins that are taste-
active. We report the three-dimensional structure of thauma-
tin I at 3.1 A resolution.
Taste is one of the least understood senses from a biochemi-
cal point of view. Sweet taste can be elicited to varying de-
grees by a wide variety of compounds in high concentration,
such as mono- and disaccharides, several amino acids, di-
peptide derivatives, glycerol, cyclamates, saccharine, ace-
sulfame, some hydrated inorganic compounds, and others.
Sensations of sweet taste are elicited by sweet compounds
binding to sweet receptors of taste buds on the tongue, simi-
lar to hormone-hormone receptor binding. However, the
similarity ends here. A 50% binding of hormone receptors
usually occurs at around 10-8 to 10-11 M concentration of
hormones, but in the case of sweet receptors the concentra-
tion to register sweet taste is about 10-1 to 10-3 M of su-
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crose. Furthermore, like other sensory perceptions, taste is
associated with a unique set of sensation parameters. These
are threshold concentration, onset time, intensity, saturation
concentration, lingering time, aftertaste, and potentiation of
other tastes or flavors. Systematic studies of these proper-
ties of taste-active compounds have been difficult in the past
because of high levels of nonspecific binding between the
sweet comnpounds and the sweet receptors due to the high
concentrations of the sweet compounds required to elicit the
sweet taste.
One of the most interesting and unusual recent findings is
the existence of two intensely sweet proteins, monellin and
thaumatin (for a recent review, see ref. 1). The sweet taste of
the proteins can be registered at a very low concentration
(10' M), comparable to those in hormone-hormone recep-
tor interaction. These proteins are about 100,000 times
sweeter than sucrose on a molar basis and several thousand
times sweeter on a weight basis. In fact, these two proteins
are the two sweetest compounds known to man. With these
proteins, studies similar to those performed for hormone-
hormone receptors can be initiated. Three-dimensional
structures of these proteins are likely to provide a solid foun-
dation upon which studies to understand the molecular basis
of sweet taste can be designed. We here report the crystal
structure of thaumatin I at 3.1 A resolution.
Thaumatins have been isolated from the fruit of a West
African rain forest shrub, Thaumatococcus daniellii Benth
(2), which has been used for centuries by inhabitants of the
region to sweeten foods such as bread and palm wine, There
are two major sweet proteins in the fruit, thaumatin I and II,
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
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1406
with almost identical molecular weights of 22,000. They con-
sist of 207 amino acids and have identical amino acid se-
quences except for five residues (3, 4). There are no histidine
residues, but there are eight disulfide bonds. Partially pure
thaumatin is available commercially from Sigma and a purifi-
cation procedure has been published (2). Although both
monellin and thaumatin are intensely sweet there is a very
limited amino acid homology between them: five regions of
thaumatin have tripeptide homologies with five regions of
monellin (3). Whether one of these homologous tripeptides
or any combination of them is responsible for the sweet taste
is not known.
Thaumatin, with many basic groups, has a pI of 12. Acyla-
tion and methylation of lysine residues and modification of
arginine residues indicate that some lysine residues are im-
portant for the sweet taste (5, 6), but the data do not identify
which basic residue or residues are important for the taste.
Antibodies raised against thaumatin have been shown by
van der Wel and Bel (7) and Hough and Edwardson (8) to
cross-react with monellin. Reciprocal experiments also show
that antibodies raised against monellin cross-react with thau-
matin (unpublished results). These results suggest structural
similarity between portions of the two sweet protein mole-
cules. Furthermore, when these proteins are bound to their
respective cross-reacting antibodies, the sweetness disap-
pears (unpublished results).
METHODS
Thaumatin I has been crystallized in two different crystalline
forms (9). Both crystalline forms are unstable in most heavy
atom soaking conditions (unpublished results). We have sub-
sequently found a new crystallization condition that made it
possible to obtain many heavy atom derivatives. The new
crystal form has the same space group as the previous one
(9) but with slightly different unit cell parameters. These
crystals diffract to 1.6 A resolution and are stable in the pres-
ence of many different heavy atom compounds that we have
tried so far. The best crystal forms are obtained from a solu-
tion containing 20 mg of protein per ml, 50 mM Hepes buffer
(pH 7.0), and 1.5 M ammonium sulfate. This solution is
equilibrated with 2.0-2.5 M ammonium sulfate through va,
por-phase equilibrium at room temperature. The crystals are
then transferred to an artificial mother liquor containing 2 M
ammonium sulfate and 50 mM Hepes buffer at pH 7. After a
week or more, they are used for data collection or heavy
atom soaking.
The space group of the crystal is P212121 with cell parame-
ters of 74.42 x 53.38 x 52.25 A. Density measurement of the
crystals indicated one protein molecule per asymmetric unit,
Abbreviation: MIR, multiple isomorphous replacement.
*Visiting Scientist from the University of Utrecht, Utrecht, The
Netherlands.
 Biochemistry: de Vos et aL Proc. NatL Acad. Sci USA 82 (1985) 1407

Table 1. Heavy atom soaking

Concentration, Time, R factor, No. of Temp. factors,
Compound mM days F sites Occupancies, e A2
KAu(CN)2 30 11 13 4 30.1, 13.3, 10.2, 13.5 49, 49, 41, 47
K2Pd(CN)4 30 2 16 3 24.5, 13.4, 14.8 24, 14, 17
K2PtI6 10 13 18 5 18.3, 14.3, 29.4, 17.4, 13.7 31, 29, 21, 23, 25
K2Pt(SCN)6 10 3 19 5 42.6, 30.6, 18.3, 10.5, 9.3 46, 53, 38, 24, 30
K2Hg(SCN)4 10 8 19 5 46.4, 17.2, 15.3, 15.5, 13.0 34, 32, 43, 36, 30
K2HgI4 0.5 7 12 4 18.5, 21.0, 19.0, 11.9 34, 32, 35, 43

with 50o of the crystal volume being solvent. X-ray diffrac-
tion data were collected at 40C on a Nicolet four-circle auto-
matic diffractometer modified with an extended detector
arm and helium-filled incident and diffracted beam collima-
tors. Ni-filtered CuK-a wavelength was used. Using an o-
step scan, diffraction intensities were measured at seven
steps spanning 0.20 in c near each diffraction center, and the
highest sum of five consecutive steps representing the flat
peak top (10) was taken to represent the integrated diffrac-
tion intensity of each reflection. Crystal decay was moni-
tored by several standard reflections, and an absorption cor-
rection was applied according to the semiempirical method
using 4i scans (11) of several reflections with X values near
900.
From many soaking experiments, six derivatives were
chosen as best for phase determination: KAu(CN)2,
K2Pd(CN)4, K2PtI6, K2Pt(SCN)6, K2Hg(SCN)4, and
K2HgI4. R factors between the native and the above six de-
rivative data sets at 3.5 A resolution are 13%, 16%, 18%,
19%, 19%, and 12%, respectively (see Table 1). The average
figure of merit after refinement by the multiple isomorphous
replacement (MIR) method is 0.78 with phasing power (rms
FH/residual) ranging between 1.4 and 2.0. A summary of the
phase refinement results is shown in Table 2.
An electron density map was calculated by using the
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were extended from 3.5 A to 3.1 A by using a program by
Wang (ref. 12; see Table 3). This new map not only resolved
a few ambiguous regions in the MIR map but also enabled us
to trace the entire backbone: almost all large side chains,
such as tryptophanes, phenylalanines, and tyrosines, could
be recognized at locations expected from the amino acid se-
quence of the protein. Furthermore, eight disulfide bonds
were clearly visible (see below).
RESULTS
The most prominent feature of the structure (see Fig. 1) is a
domain consisting of a long /3 sheet folded over into a flat-
tened "X3 barrel." Of the 11 /3 strands in this domain, each
strand is antiparallel to its neighbors with the exception of
the NH2-terminal strand and the COOH-terminal strand,
which are parallel to each other. Unlike many protein struc-
tures with 13 barrels (for a review, see ref. 13), the /3 strands
of one flat sheet are almost parallel to and on top of those of
the other sheet. Both sheets exhibit the usual right-handed
twist (13). One short and one long /3 strand at the ends con-
nect the two sheets to form the flattened /3 barrel. To this
main domain are attached two small domains rich in disul-
fide bonds. Fig. 2 shows a simplified schematic diagram
showing the arrangement of the main domain (I) and the two

phases determined by the MIR method. The map showed a attached small domain appendages (II and III) with short /3
clear molecular boundary and P structures over a large por- ribbons and disulfide bonds. Fig. 2 also shows the secondary
tion of the molecule. Although most of the backbone could structure topology of the molecules as well as the assignment
be traced into several fragments, it was impossible to trace of the disulfide bonds.
the backbone uniquely from this map. The presence of eight Our backbone structure makes a unique assignment of
disulfide bonds further complicated interpretation of the eight disulfide bonds, as shown in Fig. 2, which agrees very
map. Subsequently, the phases were improved by flattening well with that based on biochemical studies (14) except for
the solvent regions outside the molecular boundaries and the two disulfide bonds 134-145 and 149-158. The electron
Table 2. Refinement statistic for the heavy atom derivatives
Resolution, A 12.6 9.2 7.2 5.9 5.0 4.4 3.9 3.5
Reflections, no. 58 106 184 262 363 476 626 774

KAu(CN)2
R-Cullis 0.54 0.50 0.52 0.52 0.61 0.68 0.73 0.80
rms FH/residual 1.55 1.81 1.75 2.09 1.72 1.48 1.18 1.04
K2Pd(CN)4
R-Cullis 0.41 0.54 0.56 0.51 0.55 0.65 0.61 0.71
rms FH/residual 2.17 1.60 1.75 2.03 1.58 1.25 1.26 1.23
K2PtI
R-Cullis 0.51 0.37 0.44 0.55 0.51 0.57 0.57 0.65
rms FH/residual 1.90 2.00 2.32 2.24 1.92 1.90 1.50 1.52
K2Pt(SCN)6
R-Cullis 0.51 0.51 0.64 0.62 0.71 0.70 0.75 0.78
rms FH/residual 1.37 1.39 1.71 1.91 1.55 1.58 1.43 1.23
K2Hg(SCN)4
R-Cullis 0.61 0.58 0.49 0.47 0.64 0.63 0.67 0.66
rms FH/residual 1.09 1.13 1.55 1.99 1.98 2.02 1.77 1.64
K2HgI4
R-Cullis 0.53 0.45 0.46 0.49 0.47 0.62 0.68 0.70
rms FH/residual 1.99 2.79 3.17 3.27 3.08 1.77 1.26 1.38

Figure of merit 0.83 0.86 0.88 0.87 0.85 0.79 0.73 0.71
 1408 Biochemistry: de Vos et aL Proc. NatL Acad Scd USA 82 (198S)

Table 3. Density modification and phase extension I[

lilt
I* Ilt a b
Resolution, A 3.5 3.5 3.5 3.1
Reflections, no. 2614 2614 2614 1034
Figure of merit 0.78 0.89 0.90 0.68
R factor 0.22 0.24 0.27
Aa' 15 15
= S(Fob, - FC,1)2/1F2bs. Aa0 = Average accumulated phase dif-
ference.
*Original MIR data.
tAfter solvent flattening.
tAfter solvent flattening and phase extension; a = original data (to
3.5 A) and b = new data (3.5-3.1 A).

density map of these regions is clear and the assignments are

unambiguous.
Although the sweet intensities of thaumatin and monellin
are similar, there is only a very limited homology in the ami-
no acid sequences of the two proteins. As mentioned earlier,
five tripeptides in monellin have their homologous counter-
parts in thaumatin at residues 94-96, 100-102, 101-103, 118-
120, and 128-130 (3). Four of these are located in the main FIG. 2. Topological structure of thaumatin I. There are two (3
domain I and one is between domains I and II (for a stereo- sheets in the structure. The 13 strands of the top sheet are indicated
drawing of the backbone structure, see Fig. 3). All five re- by wide arrows and those of the bottom sheet, by narrow arrows.
gions are well exposed. It is likely that some of these regions Also shown are three domains of the protein and a crystallographic
are responsible for the immunological cross-reactivity be- assignment of disulfide bonds shown in black vertical bars.
tween monellin and thaumatin, and their conformation may
be important for sweet receptor binding.
ing motifs: a folded 3 sheet, or a flattened P "barrel," and
DISCUSSION the P ribbons and small loops stabilized by disulfide bonds.
The first motif is found in many other proteins (13), and the
Thaumatin and monellin are the two sweetest compounds second is found in snake venom toxin, bungarotoxin, wheat
known to man and represent a unique class of proteins that germ agglutinin, cytotoxins, and ragweed pollen allergens
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are taste-active. The thaumatin structure utilizes two build-
(15), all of which bind to membrane-bound receptors. There-
fore, it is tempting to speculate that domain II or III (or both)
may be important for binding to the membrane-bound sweet
receptors.
As mentioned earlier, antibodies raised against thaumatin
cross-react with monellin and vice versa. It will be interest-
ing to compare the crystal structures of both thaumatin and
monellin. The crystal structure of the latter awaits determi-
nation.
Recently the gene for thaumatin II has been cloned (4) and
expressed (16). Thaumatin II is as intensely sweet as thau-
matin I but is different from it at five positions [residues 46,
63, 67, 76, and 113 (4)]. It is almost certain that thaumatin II
has the same three-dimensional structure as thaumatin I de-
scribed here. The crystal structure of thaumatin may provide
an important insight as to what regions of the molecule may
need to be systematically analyzed by either site-specific
mutation or some other methods to investigate the struc-
ture-taste relationship of this protein and the associated
properties of sweet taste.

We are grateful to our colleagues, Stephen Holbrook, Craig

FIG. 1. Backbone structure of thaumatin I. The main body of the
structure consists of two ,B sheets forming a flattened ,B barrel.
Strands in the top sheet are shaded light, and those in the bottom
sheet are darker. Open bars represent disulfide bonds, and the re-
gions with sequences homologous to monellin are indicated by the
hatched marks. The viewing direction is along the crystallographic C
axis.
Ogata, David Pearlman, Gary Smith, and Everett Harvey (Universi-
ty of California, Berkeley) for various advice and help on computer
programs and hardware and to Dr. B. C. Wang (Veterans Adminis-
tration Hospital, Pittsburgh, PA) and Dr. T. N. Bhat (National Insti-
tutes of Health, Bethesda, MD) for their programs for phase im-
provement by solvent flattening. The purified thaumatin I was pro-
vided by H.v.d.W. and preliminary crystallographic studies on
thaumatin I, crystallized from a condition different from that de-
scribed here, were performed at the laboratory of H.K. and A.F.P.
The work described here has been performed at the University of
California, Berkeley, with the support of the National Institutes of
Health (NS 15174) and the Department of Energy.
 Biochemistry: de Vos et aL
FIG. 3. Stereodrawing of the backbone structure of thaumatin I. To improve the visibility of the stereodrawing, the viewing direction is
slightly tilted from the crystallographic C axis.
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