Reduced sweetness of a monellin (MNEI) mutant results from increased protein flexibility and disruption of a distant poly-(L-proline) II helix.
Reduced sweetness of a monellin (MNEI) mutant results from increased protein flexibility and disruption of a distant poly-(L-proline) II helix.
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DOI：
10.1093/chemse/bjr007
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发表时间：
2011-06
期刊：
影响因子：
3.5
通讯作者：
C. Templeton;Saeideh Ostovar pour;J. Hobbs;E. Blanch;S. Munger;G. Conn
中科院分区：
文献类型：
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作者：
C. Templeton;Saeideh Ostovar pour;J. Hobbs;E. Blanch;S. Munger;G. Conn
Monellin是一种非常有效的甜味蛋白，但人们对它如何与甜味受体相互作用知之甚少。我们测定了3种单链monellin （MNEI）蛋白的x射线晶体结构，其中2个核心残基（G16A、V37A和G16A/V37A）发生了改变，导致甜度相对于野生型蛋白降低了2至10倍。令人惊讶的是，没有观察到整体蛋白质折叠或表面氨基酸位置的变化，这对MNEI甜度很重要，可以解释这些蛋白质活性的差异。差示扫描量热法显示，虽然每个突变体MNEI的热稳定性都有所降低，但最不甜的突变体G16A-MNEI并不是最不稳定的蛋白质。相反，溶液光谱测量显示蛋白质柔韧性和c端结构的变化与蛋白质活性直接相关。G16A突变诱导的蛋白质核心紊乱是通过疏水相互作用的改变来传播的，疏水相互作用破坏了关键的c端多-（l -脯氨酸）II螺旋的形成和/或位置。这些发现表明，MNEI与甜味受体的相互作用对其蛋白质表面关键残基的相对位置高度敏感，G16A-MNEI的甜味损失可能是由于结合的熵成本增加所致。
Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding.