Department of Chemistry, University of Utah, 315 South 1400 East RM. 2020, Salt Lake City, Utah 84112, USA.
J Chem Phys. 2024 Aug 28;161(8). doi: 10.1063/5.0226075.
Fluorescent lipid probes such as 1-palmitoyl-2-(6-[7-nitro-2-1,3-benzoxadiazol-4-yl]amino-hexanoyl)-sn-glycero-3-phosphocholine (C6 NBD-PC) have been used extensively to study the kinetics of lipid flip-flop. However, the efficacy of these probes as reliable reporters of native lipid translocation has never been tested. In this study, sum-frequency vibrational spectroscopy (SFVS) was used to measure the kinetics of C6 NBD-PC lipid flip-flop and the flip-flop of native lipids in planar supported lipid bilayers. C6 NBD-PC was investigated at concentrations of 1 and 3 mol. % in both chain-matched 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chain-mismatched 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) to assess the ability of C6 NBD-PC to mimic the behavior of the surrounding matrix lipids. It was observed that C6 NBD-PC exhibited faster flip-flop kinetics compared to the native lipids in both DPPC and DSPC matrices, with notably accelerated rates in the chain-mismatched DSPC system. SFVS was also used to measure the acyl chain orientation and gauche content of C6 NBD-PC in both DPPC and DSPC membranes. In the DSPC matrix (chain mismatched), C6 NBD-PC was more disordered in terms of both gauche content and acyl tilt, whereas it maintained an orientation similar to that of the native lipids in the DPPC matrix (chain matched). In addition, the flip-flop kinetics of C6 NBD-PC were also measured using second-harmonic generation (SHG) spectroscopy, by probing the motion of the NBD chromophore directly. The flip-flop kinetics measured by SHG were consistent with those obtained from SFVS. This study also marks the first instance of phospholipid flip-flop kinetics being measured via SHG. The results of this study clearly demonstrate that C6 NBD-PC does not adequately mimic the behavior of native lipids within a membrane. These findings also highlight the significant impact of the lipid matrix on the flip-flop behavior of the fluorescently labeled lipid, C6 NBD-PC.
荧光脂质探针，如 1-棕榈酰-2-（6-[7-硝基-2-1,3-苯并恶唑-4-基]氨基-己酰基）-sn-甘油-3-磷酸胆碱（C6 NBD-PC），已被广泛用于研究脂质翻转的动力学。然而，这些探针作为天然脂质转位可靠报告者的功效从未经过测试。在这项研究中，和频振动光谱（SFVS）被用于测量 C6 NBD-PC 脂质翻转和平面支撑脂质双层中天然脂质翻转的动力学。C6 NBD-PC 以 1 和 3 mol.％的浓度在链匹配的 1,2-二棕榈酰-sn-甘油-3-磷酸胆碱（DPPC）和链不匹配的 1,2-硬脂酰-sn-甘油-3-磷酸胆碱（DSPC）中进行了研究，以评估 C6 NBD-PC 模拟周围基质脂质行为的能力。观察到 C6 NBD-PC 与 DPPC 和 DSPC 基质中的天然脂质相比，具有更快的翻转动力学，在链不匹配的 DSPC 体系中，翻转速率明显加快。SFVS 还用于测量 C6 NBD-PC 在 DPPC 和 DSPC 膜中的酰基链取向和 gauche 含量。在 DSPC 基质（链不匹配）中，C6 NBD-PC 在 gauche 含量和酰基倾斜方面都更加无序，而在 DPPC 基质（链匹配）中，它保持与天然脂质相似的取向。此外，C6 NBD-PC 的翻转动力学也通过二次谐波产生（SHG）光谱测量，通过直接探测 NBD 发色团的运动来测量。通过 SHG 测量的翻转动力学与通过 SFVS 获得的动力学一致。这项研究也是首次通过 SHG 测量磷脂翻转动力学。该研究结果清楚地表明，C6 NBD-PC 不能充分模拟膜内天然脂质的行为。这些发现还突出了脂质基质对荧光标记脂质 C6 NBD-PC 翻转行为的重大影响。
