Department of Theoretical Chemistry and Biology , KTH Royal Institute of Technology , Roslagstullsbacken 15 , S-106 91 Stockholm , Sweden.
Biomedical Research Institute , Hasselt University , Agoralaan Building C , 3590 Diepenbeek , Belgium.
Langmuir. 2018 Jul 31;34(30):9072-9084. doi: 10.1021/acs.langmuir.8b01164. Epub 2018 Jul 19.
By reverting to spectroscopy, changes in the biological environment of a fluorescent probe can be monitored and the presence of various phases of the surrounding lipid bilayer membranes can be detected. However, it is currently not always clear in which phase the probe resides. The well-known orange 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbo-cyanine perchlorate (DiI-C18(5)) fluorophore, for instance, and the new, blue BODIPY (4,4-difluoro-4-bora-3 a,4 a-diaza- s-indacene) derivative were experimentally seen to target and highlight identical parts of giant unilamellar vesicles of various compositions, comprising mixtures of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), and cholesterol (Chol). However, it was not clear which of the coexisting membrane phases were visualized (Bacalum et al., Langmuir. 2016, 32, 3495). The present study addresses this issue by utilizing large-scale molecular dynamics simulations and the z-constraint method, which allows evaluating Gibbs free-energy profiles. The current calculations give an indication why, at room temperature, both BODIPY and DiI-C18(5) probes prefer the gel (S) phase in DOPC/DPPC (2:3 molar ratio) and the liquid-ordered (L) phase in DOPC/SM/Chol (1:2:1 molar ratio) mixtures. This study highlights the important differences in orientation and location and therefore in efficiency between the probes when they are used in fluorescence microscopy to screen various lipid bilayer membrane phases. Dependent on the lipid composition, the angle between the transition-state dipole moments of both probes and the normal to the membrane is found to deviate clearly from 90°. It is seen that the DiI-C18(5) probe is located in the headgroup region of the SM/Chol mixture, in close contact with water molecules. A fluorescence anisotropy study also indicates that DiI-C18(5) gives rise to a distinctive behavior in the SM/Chol membrane compared to the other considered membranes. The latter behavior has not been seen for the studied BODIPY probe, which is located deeper in the membrane.
通过反演光谱，荧光探针的生物环境变化可以被监测到，并且可以检测到周围脂质双层膜的各个相的存在。然而，目前并不总是清楚探针位于哪个相。例如，众所周知的橙色 1,1'-十八烷基-3,3,3',3'-四甲基吲哚碳菁高氯酸盐（DiI-C18(5)）荧光团和新的蓝色 BODIPY（4,4-二氟-4-硼-3a,4a-二氮杂 s-茚并）衍生物在实验中被观察到靶向并突出各种组成的巨大单层囊泡的相同部分，包含二棕榈酰磷脂酰胆碱（DPPC）、二油酰基磷脂酰胆碱（DOPC）、神经鞘磷脂（SM）和胆固醇（Chol）的混合物。然而，不清楚可视化的是共存膜相中的哪一个（Bacalum 等人，Langmuir. 2016, 32, 3495）。本研究通过利用大规模分子动力学模拟和 z-约束方法来解决这个问题，该方法允许评估吉布斯自由能曲线。目前的计算给出了一个原因，即在室温下，BODIPY 和 DiI-C18(5)探针为什么更喜欢 DOPC/DPPC（2:3 摩尔比）中的凝胶（S）相和 DOPC/SM/Chol（1:2:1 摩尔比）混合物中的有序液体（L）相。这项研究强调了当探针在荧光显微镜中用于筛选各种脂质双层膜相时，它们在取向、位置以及因此在效率方面的重要差异。取决于脂质组成，两个探针的过渡态偶极矩与膜法线之间的夹角明显偏离 90°。可以看出，DiI-C18(5)探针位于 SM/Chol 混合物的头基区域，与水分子紧密接触。荧光各向异性研究还表明，与其他考虑的膜相比，DiI-C18(5)在 SM/Chol 膜中会引起独特的行为。对于研究的 BODIPY 探针，没有观察到这种行为，该探针位于膜的更深层。
