Masuda Tetsuya, Ohta Keisuke, Ojiro Naoko, Murata Kazuki, Mikami Bunzo, Tani Fumito, Temussi Piero Andrea, Kitabatake Naofumi
Laboratory of Food and Environmental Science, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Sci Rep. 2016 Feb 3;6:20255. doi: 10.1038/srep20255.
Thaumatin is an intensely sweet-tasting protein that elicits sweet taste at a concentration of 50 nM, a value 100,000 times larger than that of sucrose on a molar basis. Here we attempted to produce a protein with enhanced sweetness by removing negative charges on the interacting side of thaumatin with the taste receptor. We obtained a D21N mutant which, with a threshold value 31 nM is much sweeter than wild type thaumatin and, together with the Y65R mutant of single chain monellin, one of the two sweetest proteins known so far. The complex model between the T1R2-T1R3 sweet receptor and thaumatin, derived from tethered docking in the framework of the wedge model, confirmed that each of the positively charged residues critical for sweetness is close to a receptor residue of opposite charge to yield optimal electrostatic interaction. Furthermore, the distance between D21 and its possible counterpart D433 (located on the T1R2 protomer of the receptor) is safely large to avoid electrostatic repulsion but, at the same time, amenable to a closer approach if D21 is mutated into the corresponding asparagine. These findings clearly confirm the importance of electrostatic potentials in the interaction of thaumatin with the sweet receptor.
奇异果甜蛋白是一种甜度极高的蛋白质，在浓度为50 nM时就能引发甜味，按摩尔计算，该浓度是蔗糖的100000倍。在此，我们试图通过去除奇异果甜蛋白与味觉受体相互作用一侧的负电荷来生产甜度更高的蛋白质。我们获得了D21N突变体，其阈值为31 nM，比野生型奇异果甜蛋白甜得多，并且与单链莫内林的Y65R突变体一起，是迄今为止已知的两种最甜的蛋白质之一。在楔形模型框架内通过拴系对接得出的T1R2 - T1R3甜味受体与奇异果甜蛋白之间的复合物模型证实，对甜度至关重要的每个带正电荷的残基都靠近带相反电荷的受体残基，以产生最佳的静电相互作用。此外，D21与其可能对应的D433（位于受体的T1R2原体上）之间的距离足够大，可避免静电排斥，但同时，如果D21突变为相应的天冬酰胺，则便于更接近。这些发现清楚地证实了静电势在奇异果甜蛋白与甜味受体相互作用中的重要性。
