Membrane Binding analysis of select RW heptapeptides

Data from quartz crystal microbalance experiments used to analyse binding to lipid membranes of RW heptapeptides. <br>This folder contains both the raw data files and list of extracted binding values.<br>Phospholipid 1,2-dioleoyl-sn-glycero-phosphocholin (DOPC) and 1,2-dioleoyl-<i>sn</i>-glycero-3-phospho-<i>rac</i>-(1-glycerol) (DOPG) were obtained from Avanti Polar Lipids Inc (Alabama, USA). Neutral lipid bilayers were formed from vesicles composed entirely of DOPC, whilst anionic bilayers were composed of DOPC and DOPG at a ratio of 4:1 respectively. Lipids were dissolved in chloroform in a glass vial and then dried to a film under nitrogen gas. The lipids were then resuspended in HEPES buffer with 150 mM NaCl at 1 mg/ml and frozen at -80 °C for 30 minutes. This suspension was thawed, before sonication at 10 Hz in 6 to 10 bursts, each lasting 1 minute (MSE Soniprep 150 plus; MSE Centrifuges Heathfield, UK). The size of the resulting vesicles was assessed using DLS to verify the vesicles were of a target diameter of 30 nm (Zetasizer NanoS; Malvern Instruments Ltd. Malvern, UK.) and the suspension was stored in 200 µl aliquots 4 °C and used for experiments within two weeks of preparation.<br>Membrane-peptide interactions were assessed using a quartz crystal microbalance with energy dissipation monitoring (QCMD) (Q-Sense Omega; Biolin Scientific. Manchester, UK); a subset of heptapeptides (n=31) was analysed, which form the 21 pairs of peptides that had the largest differences in IC50 values whilst differing by only a single substitution in the peptide sequence. The stock vesicle suspension was diluted 1 in 5 with HEPES, and then 10 µl of 1 M CaCl₂ was added. SiO₂ sensors were cleaned using UV/Ozone (Bioforce Nano Procleaner) and lipid bilayers were prepared by depositing 0.2 mg/ml vesicle suspension at a rate of 25 µl/minute for 5 min, followed by removal of excess lipid by adding HEPES buffer onto the chip at 25 µl/minute for 5 minutes. The frequency shift of the quartz chip was monitored throughout to ensure that the lipid bilayer had formed correctly with a frequency of -25 Hz. Once a stable bilayer had been formed, peptide solutions at 20 µM (chosen due to its location centrally within the IC50 distributions of the peptides) in HEPES buffer were deposited onto the bilayer at a rate of 20 µl/minute for 9 minutes. Next, HEPES buffer was added at a rate of 25 µl/minute for 5 minutes, to remove any weakly bound or unbound peptide. Frequency and energy dissipation changes were recorded using Q-soft and the frequency shift in the 5^th octave was converted to mass shift using Q-Tools software (Q-sense Omega, BiolinScientific/ Q-Sense, Sweden). The mass change between the stable bilayer and the end of the final washing step was then used to determine the amount of peptide bound to the membrane. The R package<i> Changepoints</i> (version 2.2.2) was used to determine the average mass of peptide bound to the bilayer at each step. 2 independent experiments were performed with two technical replicates for each.

本数据集来源于石英晶体微天平实验数据，用于分析RW七肽与脂质膜的结合特性。
本文件夹同时包含原始数据文件与提取得到的结合值列表。
所用磷脂1,2-二油酰-sn-甘油-磷酸胆碱（1,2-dioleoyl-sn-glycero-phosphocholin，DOPC）与1,2-二油酰-sn-甘油-3-磷酸-rac-(1-甘油)（1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)，DOPG）均购自美国阿拉巴马州Avanti Polar Lipids公司。中性脂质双层由纯DOPC囊泡制备，而阴离子脂质双层则由DOPC与DOPG按4:1的摩尔比混合制备。将脂质置于玻璃小瓶中溶于氯仿，随后在氮气吹扫下干燥成薄膜。之后将脂质重悬于含150 mM氯化钠的HEPES缓冲液中，浓度为1 mg/ml，于-80℃冷冻30分钟。将悬浮液解冻后，使用超声仪以10 Hz频率进行6~10次脉冲超声，每次持续1分钟（型号：MSE Soniprep 150 plus，制造商：英国希思菲尔德MSE离心机公司）。使用动态光散射（dynamic light scattering，DLS）对制备得到的囊泡粒径进行表征，确认其目标粒径为30 nm（仪器型号：Zetasizer NanoS，制造商：英国马尔文仪器有限公司）。将悬浮液以200 μl为单位分装，于4℃储存，并于制备后两周内用于实验。
使用石英晶体微天平-耗散监测（quartz crystal microbalance with energy dissipation monitoring，QCMD）系统（型号：Q-Sense Omega，制造商：英国曼彻斯特Biolin Scientific公司）表征膜-肽相互作用；本次实验分析了31个七肽子集，对应21对肽段，这些肽段的半数抑制浓度（IC50）差异最大，且仅存在单个氨基酸取代。将储存的囊泡悬浮液用HEPES缓冲液按1:5比例稀释，随后加入10 μl 1 M氯化钙溶液。使用紫外臭氧清洗仪（Bioforce Nano Procleaner）对二氧化硅（SiO₂）传感器进行清洗，随后以25 μl/min的流速将0.2 mg/ml的囊泡悬浮液沉积5分钟以制备脂质双层，之后以25 μl/min的流速向芯片表面加入HEPES缓冲液冲洗5分钟以去除多余脂质。全程监测石英芯片的频率偏移，确保脂质双层正确形成，目标频率偏移为-25 Hz。待脂质双层稳定后，以20 μl/min的流速将浓度为20 μM的肽溶液（该浓度选取依据为其处于所有肽段IC50分布的中间位置）沉积在双层表面9分钟。随后以25 μl/min的流速加入HEPES缓冲液冲洗5分钟，以去除弱结合或未结合的肽段。使用Q-soft软件记录频率与能量耗散的变化，并使用Q-Tools软件（型号：Q-sense Omega，制造商：瑞典BiolinScientific/Q-Sense公司）将第5倍频程的频率偏移转换为质量偏移。以稳定脂质双层与最终冲洗步骤结束时的质量变化量，计算结合至膜上的肽段总量。使用R语言包Changepoints（版本2.2.2）计算每一步骤中结合至脂质双层的肽段平均质量。本次实验共开展2次独立重复，每次独立实验包含2次技术重复。