SUPA School of Physics and Astronomy, University of St. Andrews, North Haugh, Fife, KY16 9SS, UK.
Institute of Biological Physics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, EH14 4AS, UK.
Sci Rep. 2019 Sep 9;9(1):12897. doi: 10.1038/s41598-019-49210-0.
The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.
通过去污剂溶解膜对于许多技术应用至关重要，并且在生物化学研究中已广泛用于诱导细胞破裂、提取细胞成分，以及纯化、重建和结晶膜蛋白。去污剂溶解的热力学细节已得到广泛研究，但动力学方面仍知之甚少。在这里，我们使用了单个囊泡荧光共振能量转移（svFRET）、荧光相关光谱和石英晶体微天平耗散监测的组合，以获得亚微米大小囊泡的实时动力学和基本溶解步骤，这些步骤是传统的衍射受限光学方法无法实现的。实时注入非离子去污剂 Triton X 会诱导用 Dil/DiD FRET 对标记的表面固定囊泡的两相溶解动力学。通过 svFRET 实现的纳米级灵敏度使我们能够将每个动力学步骤明确分配给囊泡结构的变形，包括初始快速囊泡肿胀事件，随后是缓慢的脂质损失和胶束化。我们预计 svFRET 平台将适用于此处研究的亚微米尺寸以外的范围，并成为揭示去污剂-脂质相互作用复杂动力学的独特工具。
