Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA.
Biophys J. 2013 Jun 4;104(11):2410-8. doi: 10.1016/j.bpj.2013.04.039.
Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs--cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)--on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl⁺). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl⁺ influx-the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate.
小分子光稳定剂或保护剂 (PAs) 为单分子光谱和荧光显微镜中荧光染料的稳定性需求提供了重要支持。这些试剂也用于细胞膜和模拟脂质双层环境的模型系统的研究，但关于它们对膜结构和物理性质的可能影响的信息很少。鉴于两亲性小分子对双层性质（如弹性和固有曲率）的影响，我们研究了六种常用的 PA——环辛四烯 (COT)、对硝基苄醇 (NBA)、Trolox (TX)、1,4-二氮杂二环[2.2.2]辛烷 (DABCO)、对硝基苯甲酸 (pNBA) 和没食子酸丙酯 (nPG)——对双层性质的影响，方法是使用基于短杆菌肽 A (gA) 的荧光猝灭测定法来探测 PA 诱导的 gA 单体↔二聚体平衡变化。实验使用荧光染料负载的大单层囊泡进行，该囊泡中掺杂了 gA，并使用 gA 通道可渗透的荧光猝灭剂 (Tl⁺) 测定 gA 单体↔二聚体平衡的变化。例如，PA 在双层/溶液界面吸附会改变 gA 通道形成的平衡常数，从而改变大单层囊泡膜中导电 gA 通道的数量，这将导致 Tl⁺内流率（荧光猝灭率）发生变化，从而改变双层性质。在实验相关的毫摩尔浓度范围内，TX、NBA 和 pNBA 引起 gA 通道活性的可比增加。在毫摩尔范围内的 COT 也导致 gA 通道活性略有降低。nPG 在亚毫摩尔浓度下增加了通道活性。DABCO 没有改变 gA 活性。在实验相关浓度下，六种测试的 PA 中有五种改变了脂质双层性质，这对于设计和分析细胞和模型膜系统中的荧光研究变得很重要。因此，我们测试了 COT、NBA 和 TX 的组合；与假设它们对双层性质的影响具有加性相比，这些组合改变荧光猝灭率的程度较小。COT 和 NBA 等摩尔浓度的组合导致荧光猝灭率的变化最小。
