Brazzein is the smallest, most stable, member of a family of sweet-tasting proteins. As such Brazzein presents intriguing possibilities for both scientific research and commercial applications. Its unusual thermostability (activity is retained after 2 hours at 90xC) and high potency (500 times sweeter than sucrose on a weight basis) render it a potential candidate for commercial use as a versatile low calorie sweetener. Its other physical properties, wide range of pH stability (from pH 3 to 8) and small size render it an excellent candidate for NMR studies. NMR spectra (COSY, HOHAHA, NOESY) have been acquired at 500 and 600 MHz proton frequency. The homonuclear 2D NMR resonances of brazzein spin systems have been completely assigned to all 54 residues in the amino acid sequence. A preliminary structure has been calculated by distance geometry, using constraints derived from 1H-1H NOE crosspeak intensities and 3JHNHf coupling constants. This structure reveals that brazzein contains a helix-turn-helix motif, which is cradled by a three-stranded beta sheet. This topology has been supported by chemical shift calculations, hydrogen exchange data, and coupling constant information. A higher resolution structure will emerge as the existing distance constraints are refined. Additional NMR experiments are currently being carried out to obtain data for a more precise, higher resolution, structure.
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