Péterffy Judit Petres, Szabó Mária, Szilágyi László, Lányi Szabolcs, Ábrahám Beáta
Department of Inorganic Substances Technology and Environment, Protection, "Politechnica" University of Bucharest, Polizu street No. 1-7, 011061, Bucharest, Romania,
J Fluoresc. 2015 Jul;25(4):871-83. doi: 10.1007/s10895-015-1567-4. Epub 2015 Apr 19.
The histidine-modified EGFP was characterized as a sensing element that preferentially binds nanomolar concentrations of Cu(2+) in a reversible manner (Kd = 15 nM). This research aims to determine the causes of nanomolar-affinity of this mutant by investigating significant structural and energetic alterations of the chromophore in the presence of different copper ion concentrations. In order to reveal the unknown parts of the quenching mechanism we have elaborated a specific approach that combines theoretical and experimental techniques. The theoretical experiment included the modeling of potential distortions of the chromophores and the corresponding changes in energy using quantum mechanical calculations. Differences between the modeled energy profiles of planar and distorted conformations represented the energies of activation for the chromophore distortions. We found that some values of the experimental activation energies, which were derived from fluorescence lifetime decay analysis (ex: 470 nm, em: 507 nm), were consistent with the theoretical ones. Thus, it has been revealed similarity between the theoretical activation energy (50 kJmol(-1)) for 40° phenolate-ring distortion and the experimental activation energy (52.17 kJmol(-1)) required for histidine-modified EGFP saturation with copper. This chromophore conformation was further investigated and it has been found that the large decrease in fluorescence emission is attributed to the significant charge transfer over the molecule which triggers proton transfer thereby neutralizing the cromophore.
组氨酸修饰的增强型绿色荧光蛋白（EGFP）被表征为一种传感元件，它以可逆方式优先结合纳摩尔浓度的Cu(2+)（解离常数Kd = 15 nM）。本研究旨在通过研究在不同铜离子浓度下生色团显著的结构和能量变化，来确定该突变体具有纳摩尔亲和力的原因。为了揭示猝灭机制中未知的部分，我们精心设计了一种结合理论和实验技术的特定方法。理论实验包括使用量子力学计算对生色团的潜在畸变及其相应的能量变化进行建模。平面构象和畸变构象的建模能量分布之间的差异代表了生色团畸变的活化能。我们发现，通过荧光寿命衰减分析得出的一些实验活化能值（例如：激发波长470 nm，发射波长507 nm）与理论值一致。因此，已揭示出40°酚盐环畸变的理论活化能（50 kJmol(-1)）与组氨酸修饰的EGFP被铜饱和所需的实验活化能（52.17 kJmol(-1)）之间的相似性。对这种生色团构象进行了进一步研究，发现荧光发射的大幅下降归因于分子上显著的电荷转移，这引发了质子转移，从而使生色团中和。
