THE JOURNAI. OF BIOLOCICAI. CHEMISTRY Vol. 2%. No. 23. Iswe of 1)eremher I O . pp. 1247ti-I?477. I W I Prrnt~d in I'.S.A.

Preliminary Crystallographic Studies of a Sweet Protein, Monellin" (Received for publication, May 26, 1981)

Gail E. TomlinsonSg and Sung-Hou Kim1 From the $ Department of Biochemistry, Duke Unicersity, Durham, North Carolina 27710 and the 7 Department of Chemistry, Universit-v of California at Berkeley, Berkeley, California 94720

Monellin belongs to a new class of proteins that have intensely sweet taste. We report here the results of crystallization of this protein and crystallographic pa- rameters of the monellin crystals: space group is P2,; the cell parameters are 39.1 X 71.5 X 86.9 A with = 107.6"; and there are 4 monellin molecules/asymmetric unit.

It has been known for some time that a sweet stimulus may be elicited by a number of compounds, including several mono- and disaccharides, several amino acids and dipeptides, glycerol, chloroform, the well known cyclamates and sac- charin, as well as several hydrated inorganic compounds. These substances come from basically very different classes of compounds, and it is difficult to identify the common chemical and/or structural properties that may be responsible for the sweet taste, especially since many of these molecules presumably can assume varieties of conformation in response to their environment.

Recently, a totally new class of sweet compounds has been isolated from various tropical berries, and shown to be proteins (1). Two of these proteins, monellin and thaumatin, elicit a sweet sensation, which is roughly 100,000 times as intense as that of sucrose when compared on a molar basis (2) and more than 3,000 times as intense when compared on a milligram basis. Monellin and thaumatin are both rather small proteins having molecular weights of 10,700 and 21,000, respectively, and contain no carbohydrate. Monellin is composed of two nonidentical subunits ( 3 ) , while thaumatin is composed of a single polypeptide chain (4).

Tertiary structures of both monellin and thaumatin are essential for the taste effect to take place as demonstrated by circular dichroism studies employing a variety of denaturing conditions (5). In addition, the presence of both subunits of monellin is necessary for the sweet taste to occur. The exact role of the tertiary structure in chemoreception remains un- known at the present time. Thaumatin and monellin show a similarity in sweetness intensity on a molar basis, and thus, it has been hypothesized that these two proteins have a similar taste active site. Further studies are needed, however, in order to compare the tertiary structural features among the taste- active proteins and to see if structural similarities exist be- tween these proteins and the smaller sweet compounds.

Thaumatin has been crystallized (6) and search for heavy atom derivatives is in progress.' Monellin has also been crys-

This work has been supported by a grant from the National Institutes of Health (NS 15174). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adtsertisement" in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Present address. George Washington University. School of Med- icine and Health Sciences, Washington. D.C. 20037.

' H. van der Wel. personal communication.

FIG. 1. Monellin crystal and diffraction pattern. a, single crys- t a l of monellin grown by the vapor diffusion method. About 4 mg/ml of protein in 10 mM phosphate buffer (pH 7.2) was equilibrated at 4 "C with 33.3%, (w/w) solution of polyethyleneglycol. The smallest dimension of this crystal is about 300 pm. h, the precession photo- graphs of Okl zone. The horizontal k axis shows the systematic absence of odd index reflections.

tallized (7) in an orthorhombic crystal form, but structural studies have not been pursued.2 The amino acid sequence of monellin is already known (3 , 8, 9).

We have recently crystallized monellin in a monoclinic crystal form (Fig. l a ) that is stable and diffracts x-rays very well (Fig. lb). The crystals have been obtained by a vapor diffusion method (10) from a solution containing 4 mg/ml of protein, 15% (w/w) polyethyleneglycol, 10 mM phosphate buffer (pH 7.2), which was equilibrated with 33.3% (w/w) polyethyleneglycol (average molecular weight of 6000) at 4 "C. Large well shaped crystals usually appear in 10 days. The

A. Wlodawar and K. Hodgson, personal communication.

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Preliminary Crystallographic Studies of Monellin 12477

resolution extends out to 1.9 8. The space oup is P2, with unit cell parameters of a = 39.1 A, b = 71.5 f c = 86.9 A, and /? = 107.6'. The density of the crystal (1.18 g/cm3) indicates that there are 4 monellin molecules/asymmetric unit, V, is 2.76 A"/dalton, and volume fraction of the solvent is 55%. X- ray diffraction data for the native crystals and several crystals of potential heavy atom derivatives have been collected by an automatic four-circle diffractometer and the crystallographic analysis is in progress.

Recent competition experiments using antibody raised against thaumatin suggest that sweet compounds such as saccharin, L-aspartyl-L-phenylalanyl methyl ester, sucrose de- rivatives, cyclamate, neohesperidin dihydrochalcone may have antigenic determinants common to thaumatin and mo- nellin (11, 12). The three-dimensional structure of monellin and thaumatin may provide structural information about what this common feature may be that is responsible for eliciting the intense sweet taste.

REFERENCES

1. Cagan, R. H. (1973) Science 181,32-35 2. Van der Wel, H., and Loeve, K. (1973) FEBS Lett. 29, 181-184 3. Bohak, Z., and Li, S. (1976) Biochim. Biophys. Acta 427, 153-170 4. Iyengar, R. B., Smits, P., van der Ouderaa, F., van der Wel, H.,

van Brauwershaven, J., Ravestein, P., Richters, G., and van Wasenaar, P. D. (1979) Eur. J . Biochem. 96, 193-204

5. Jirgenson, B. (1976) Biochim. Biophys. Acta 446, 255-261 6. Van der Wel, H., van Soest, T. C., and Royers, E. C. (1975) FEBS

7. Wlodawer, A., and Hodgson, K. (1975) Proc. Natl. Acad. Sci. U.

8. Hudson, G., and Biemann, K. (1976)Biochirn. Biophys. Res. Com-

9. Frank, G., and Zuber, H. (1976) Hoppe-Seyler's Z. Physiol. Chem.

10. Kim, S. H., and Quigley, G. J. (1978) Methods Enzymol. 59, 3-21 11. Hough, C. A., and Edwardson, J. A. (1978) Nature 271,381-383 12. Van der Wel, H., and Bel, W.J. (1978) Chem. Senses Flavor 3,

Lett. 56,316-317

S. A. 72,398-399

mun. 71,212-220

357,585-592

99-104