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.