Biomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Sciences, Chung-Ang University, 221, Huksuk-Dong, Dongjak-Ku, Seoul 156-756, Republic of Korea.
Food Chem. 2013 Jun 1;138(2-3):1370-3. doi: 10.1016/j.foodchem.2012.10.140. Epub 2012 Nov 10.
We have previously identified critical residues important for sweetness of the sweet protein brazzein by site-directed mutagenesis (Yoon, Kong, Jo, & Kong, 2011). In order to elucidate the interaction mechanisms of brazzein with the sweet taste receptor, we made multiple mutations of three residues (His31 in loop 30-33, Glu36 in β-strand III, and Glu41 in loop 40-43). We found that all double mutations (H31R/E36D, H31R/E41A and E36D/E41A) made the molecules sweeter than des-pE1M-brazzein and three single mutants. Moreover, the triple mutation (H31R/E36D/E41A) made the molecule significantly sweeter than three double mutants. These results strongly support the hypothesis that brazzein binds to the multisite surface of the sweet taste receptor. Our findings also suggest that mutations reducing the overall negative charge and/or increasing the positive charge favour sweet-tasting protein potency.
我们之前通过定点突变（Yoon、Kong、Jo 和 Kong，2011）鉴定了与甜蛋白 Brazzein 甜度相关的关键残基。为了阐明 Brazzein 与甜味受体的相互作用机制，我们对三个残基（loop 30-33 中的 His31、β-strand III 中的 Glu36 和 loop 40-43 中的 Glu41）进行了多次突变。我们发现所有双突变（H31R/E36D、H31R/E41A 和 E36D/E41A）都使分子比 des-pE1M-brazzein 和三个单突变体更甜。此外，三重突变（H31R/E36D/E41A）使分子比三个双突变体明显更甜。这些结果强烈支持 Brazzein 与甜味受体多结合位点结合的假设。我们的发现还表明，降低整体负电荷和/或增加正电荷的突变有利于甜蛋白的效力。
