Conformational Changes of the Sweet Protein Monellin as Measured by Fluorescence Emission

Abstract

Monellin is a protein that tastes sweet. In the native state it is a dimer composed of two dissimilar noncovalently associated polypeptides. The conformation of the protein is a determinant of its sweetness, and the present investigation takes advantage of the fluorescence spectrum being a sensitive index of its conformation. The emission spectrum is used to evaluate the ability of temperature and pH to alter the conformation and the sweetness of the protein. When monellin dissolved in water is heated in discrete steps from 25 to 100°C, its sweetness decreases. The halfwidth of the fluorescence emission band increases in parallel with the loss of sweetness. The increase in halfwidth is due primarily to an increase in the intensity of tyrosine emission that may be the result of the two dissimilar polypeptides of monellin beginning to separate. Tyrosine residues are present in both chains, while the single tryptophan occurs in only one. Monellin is less susceptible to denaturation by increasing temperature when dissolved in sodium acetate buffer at pH 4 than it is at pH 3 or 7. When the pH of a solution containing monellin in 0.1 M KCl is varied from 2 to 13, there is a broad pH range (pH 4 to 9) where monellin's conformation is not markedly altered. Below pH 3.5 and above pH 10.5, however, the emission spectra indicate that substantial denaturation occurs. However, monellin can be partially renatured following pH 12 treatment with only minimal loss of sweetness. The sweetness of monellin under these two types of denaturing conditions, temperature and pH, can be predicted by the fluorescence emission spectrum of the protein. In addition, this study confirms that the biological activity of monellin, its sweetness, is a function of quaternary structure of the protein.