Membrane Binding analysis of select RW heptapeptides

Data from quartz crystal microbalance experiments used to analyse binding to lipid membranes of RW heptapeptides. This folder contains both the raw data files and list of extracted binding values. Phospholipid 1,2-dioleoyl-sn-glycero-phosphocholin (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-rac-(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. 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 5th 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 Changepoints (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.本数据集源自用于分析RW七肽与脂质膜结合行为的石英晶体微天平实验。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。 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）购自美国阿拉巴马州的Avanti Polar Lipids Inc公司。中性脂质双层由纯DOPC组成的囊泡制备得到，而阴离子脂质双层则由DOPC与DOPG以4:1的摩尔比混合制备。将脂质溶解于玻璃小瓶的氯仿中，随后在氮气氛围下干燥成膜。接着将脂质重悬于含150 mM氯化钠的HEPES缓冲液中，浓度为1 mg/ml，随后置于-80℃冷冻30分钟。将悬浮液解冻后，使用MSE Soniprep 150 plus型超声仪（英国希思菲尔德MSE离心机公司）以10 Hz的频率进行6至10次脉冲超声，每次时长1分钟。使用动态光散射（Dynamic Light Scattering, DLS）对所得囊泡的粒径进行表征，以确认其目标粒径为30 nm（使用英国马尔文市Malvern Instruments Ltd公司的Zetasizer NanoS型粒径分析仪）。将悬浮液分装为200 μl的等份，于4℃储存，并在制备后两周内用于实验。 使用带能量耗散监测的石英晶体微天平（quartz crystal microbalance with energy dissipation monitoring, QCMD）（英国曼彻斯特Biolin Scientific公司的Q-Sense Omega型仪器）评估膜-肽相互作用。本次实验分析了31个七肽的子集，这些七肽对应21对肽段，它们的半数最大抑制浓度（IC50）值差异最大，且肽序列仅存在单个氨基酸取代。将囊泡储备悬浮液以1:5的比例用HEPES缓冲液稀释，随后加入10 μl的1 M氯化钙溶液。使用Bioforce Nano Procleaner型UV/Ozone清洗仪对二氧化硅传感器进行清洗，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，随后以25 μl/min的流速加入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，以确认脂质双层成功形成，目标频率偏移为-25 Hz。待稳定的脂质双层形成后，将浓度为20 μM的肽溶液（该浓度处于各肽段IC50分布的中心位置，故选用）以20 μl/min的流速沉积于双层表面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（瑞典Q-Sense/BiolinScientific公司的Q-sense Omega配套软件）将第5泛音的频率偏移转换为质量偏移。通过稳定脂质双层阶段与最终冲洗步骤结束时的质量变化，确定结合于膜上的肽质量。使用R包Changepoints（版本2.2.2）计算各步骤中结合于脂质双层的肽平均质量。本实验共进行2次独立重复，每次独立实验包含2次技术重复。