Hydrogen atoms are critical to the nature and properties of proteins, and thus deuteration has the potential to influence protein function. In fact, it has been reported that some deuterated proteins show different physical and chemical properties to their protiated counterparts. Consequently, it is important to investigate protonation states around the active site when using deuterated proteins. Here, hydrogen isotope effects on the S65T/F99S/M153T/V163A variant of green fluorescent protein (GFP), in which the deprotonated B form is dominant at pH 8.5, were investigated. The pH/pD dependence of the absorption and fluorescence spectra indicates that the protonation state of the chromophore is the same in protiated GFP in H₂O and protiated GFP in D₂O at pH/pD 8.5, while the pK_a of the chromophore became higher in D₂O. Indeed, X‐ray crystallographic analyses at sub‐ångström resolution revealed no apparent changes in the protonation state of the chromophore between the two samples. However, detailed comparisons of the hydrogen OMIT maps revealed that the protonation state of His148 in the vicinity of the chromophore differed between the two samples. This indicates that protonation states around the active site should be carefully adjusted to be the same as those of the protiated protein when neutron crystallographic analyses of proteins are performed.

中文翻译：

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X射线晶体学研究了亚ngström分辨率下绿色荧光蛋白的氢同位素效应。

氢原子对蛋白质的性质和特性至关重要，因此氘代可能会影响蛋白质的功能。实际上，据报道，某些氘化蛋白质与其经精制的对应蛋白质显示出不同的物理和化学性质。因此，使用氘化蛋白质时研究活性位点附近的质子化状态非常重要。在这里，研究了氢同位素对绿色荧光蛋白（GFP）的S65T / F99S / M153T / V163A变体的影响，其中去质子化的B形式在pH值为8.5时占优势。pH / pD对吸收光谱和荧光光谱的依赖性表明，在pH / pD 8.5的条件下，发色的质子状态在H ₂ O中的带蛋白GFP和D ₂ O中的带蛋白GFP是相同的，而p发色团的K _a在D ₂ O中变得更高。确实，亚ngström分辨率的X射线晶体学分析显示，两个样品之间发色团的质子化状态没有明显变化。但是，氢OMIT图的详细比较显示，两个样品在发色团附近的His148的质子化状态不同。这表明在进行蛋白质的中子晶体学分析时，应仔细调节活性位点周围的质子化状态，使其与被纯化蛋白质的质子化状态相同。