Department of Biotechnology (Biology VI), RWTH Aachen University, Aachen, Germany.
Biomed Res Int. 2013;2013:329087. doi: 10.1155/2013/329087. Epub 2013 Jul 10.
An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a "pure" PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1:10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to "wrap" the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.
与 PIB1000-PEG6000-PIB1000 囊泡表面相互作用的 EGFP 构建体报告了约 2 倍的荧光发射增强。由于该构建体具有插入 PIB 核中的两亲肽，因此预计 EGFP 将经历“纯”PEG 环境。为了揭示这一现象，使用了不同分子量和浓度的 PEG/水溶液。在约 1:10 的蛋白/PEG 摩尔比下，就观察到了荧光发射的增加，达到了与 PEG 分子量相关的平台。在甘油水溶液中进行的平行实验确实显示出荧光略有增强，但起始浓度要高得多。在纯水中、甘油中和 PEG 水溶液中进行 EGFP 的分子动力学模拟表明，PEG 分子倾向于“包裹”蛋白质，从而在局部 PEG 浓度比其本体浓度高的微环境中。由于周围蛋白质的折射率会干扰荧光发射，PEG 分子的聚集即使在极低的浓度下也会引起荧光发射增强。当涉及到分子生物学实验中 EGFP 的使用时，这些发现可能很重要。
