Institute of Atomic and Molecular Sciences (IAMS) , Academia Sinica , Taipei 10617 , Taiwan.
J Phys Chem B. 2019 Aug 1;123(30):6492-6504. doi: 10.1021/acs.jpcb.9b03789. Epub 2019 Jul 23.
Native cell-membrane-derived supported lipid bilayers (SLBs) are an emerging platform with broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is the proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact between the bilayer membrane and the supporting substrate, and thus, the mobility of the transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins with distinct structures, i.e., a GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescent protein (GFP), and their dynamics were measured by fluorescent single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length on protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1000 to 5000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were augmented with the polymer length, whereas the diffusion coefficient of the GPI-anchored protein remained almost identical for different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm/s approximately) for the cushioned membrane with the longest polymer length (molecular weight of 5000), indicating that at the microscopic length scale, the SLBs were fully suspended from the substrate by the polymer cushion. Transient confinements were observed for all three proteins, and increasing the polymer length reduced the tendency of transient confinement. The measured dynamics of membrane proteins were found to be nearly unchanged after the depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer-cushioned plasma membrane bilayers that are potentially useful for the future developments of membrane-based biosensors and analytical assays.
天然细胞膜衍生的支撑脂质双层（SLB）是一个新兴的平台，具有广泛的应用，从基础研究到下一代生物传感器。该平台的成功关键在于适当容纳膜蛋白，以保持其动力学和功能。聚合物垫通常用于避免双层膜与支撑基底直接接触，从而保持跨膜蛋白的流动性。然而，对于聚合物垫如何影响膜分子的绝对流动性知之甚少。在这里，我们描述了源自细胞膜的聚合物缓冲脂质双层中单膜蛋白的动力学，并研究了聚合物长度的影响。三种具有不同结构的膜蛋白，即糖基磷脂酰肌醇锚定蛋白、单次跨膜蛋白 CD98 重链和七次跨膜蛋白 SSTR3，与绿色荧光蛋白（GFP）融合，并通过荧光单分子跟踪测量其动力学。我们实施了自动数据采集，以用大量统计数据研究聚合物长度对蛋白质动力学的影响。我们的数据表明，增加 PEG 聚合物长度（分子量从 1000 增加到 5000）会增加膜蛋白的可移动分数。此外，随着聚合物长度的增加，跨膜蛋白的扩散系数也增加，而糖基磷脂酰肌醇锚定蛋白的扩散系数对于不同的聚合物长度几乎保持不变。重要的是，对于具有最长聚合物长度（分子量为 5000）的缓冲膜，三种膜蛋白的扩散系数变得相同（约 2.5 μm/s），这表明在微观长度尺度上，SLB 完全由聚合物垫从基底悬浮。对于所有三种蛋白质都观察到了瞬时约束，并且增加聚合物长度会降低瞬时约束的趋势。在胆固醇耗尽后，膜蛋白的测量动力学几乎保持不变，这表明观察到的固定化和瞬时约束不是由于富含胆固醇的膜纳米区（脂筏）引起的。我们的单分子动力学阐明了聚合物缓冲质膜双层的生物物理特性，这对于基于膜的生物传感器和分析测定的未来发展可能是有用的。
