do Canto António M T M, Robalo João R, Santos Patrícia D, Carvalho Alfredo J Palace, Ramalho J P Prates, Loura Luís M S
Centro de Química de Évora e Departamento de Química, Escola de Ciências e Tecnologia, Colégio Luís Verney, Rua Romão Ramalho 59, P-7002-554 Évora, Portugal.
Centro de Química de Évora e Departamento de Química, Escola de Ciências e Tecnologia, Colégio Luís Verney, Rua Romão Ramalho 59, P-7002-554 Évora, Portugal; Theory and Bio-Systems Department, Max Planck Institute of Colloids and Interfaces, Wissenschaftspark Golm, D-14424 Potsdam, Germany.
Biochim Biophys Acta. 2016 Nov;1858(11):2647-2661. doi: 10.1016/j.bbamem.2016.07.013. Epub 2016 Jul 27.
Fluorescence spectroscopy and microscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in these membranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only ~3-4Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using them as membrane reporters.
几十年来，荧光光谱法和显微镜技术一直被用作膜生物物理学的工具。由于磷脂是非荧光性的，因此在这种情况下使用外在膜探针很常见。在后者中，1,6-二苯基己三烯（DPH）及其三甲基铵衍生物（TMA-DPH）已被广泛使用。人们普遍认为，由于其额外的带电基团，TMA-DPH锚定在脂质/水界面，并报告与疏水性DPH不同的双层区域。在本研究中，我们采用原子尺度的分子动力学模拟来表征DPH和TMA-DPH在1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）和POPC/胆固醇（4:1）双层膜中的行为。我们表明，尽管TMA-DPH在这些膜中的动力学明显比DPH更受阻碍，但TMA-DPH平均荧光团的位置仅比DPH浅约3-4埃。与DPH相比，TMA-DPH在平移和旋转运动中观察到的阻碍主要不是由于深度上的显著差异，而是由于前者与带负电的脂质原子的有利静电相互作用。通过揭示对这两种探针行为的详细见解，我们的结果对于解释过去的工作以及规划未来使用它们作为膜报告分子的实验都很有用。
