Study of the Interaction of a Novel Semi-Synthetic Peptide with Model Lipid Membranes

Most linear peptides directly interact with membranes, but the mechanisms of interaction are far from being completely understood. Here, we present an investigation of the membrane interactions of a designed peptide containing a non-natural, synthetic amino acid. We selected a nonapeptide that is reported to interact with phospholipid membranes, ALYLAIRKR, abbreviated as ALY. We designed a modified peptide (azoALY) by substituting the tyrosine residue of ALY with an antimicrobial azobenzene-bearing amino acid. Both of the peptides were examined for their ability to interact with model membranes, assessing the penetration of phospholipid monolayers, and leakage across the bilayer of large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs). The latter was performed in a microfluidic device in order to study the kinetics of leakage of entrapped calcein from the vesicles at the single vesicle level. Both types of vesicles were prepared from a 9:1 (mol/mol) mixture of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1′-rac-glycerol). Calcein leakage from the vesicles was more pronounced at a low concentration in the case of azoALY than for ALY. Increased vesicle membrane disturbance in the presence of azoALY was also evident from an enzymatic assay with LUVs and entrapped horseradish peroxidase. Molecular dynamics simulations of ALY and azoALY in an anionic POPC/POPG model bilayer showed that ALY peptide only interacts with the lipid head groups. In contrast, azoALY penetrates the hydrophobic core of the bilayers causing a stronger membrane perturbation as compared to ALY, in qualitative agreement with the experimental results from the leakage assays.


Separation of Non-Entrapped HRPC from HRPC-Containing Vesicles
The non-entrapped enzyme molecules were separated from the enzyme-containing vesicles by size-exclusion chromatography using a 2 × 20 cm glass column filled with Sepharose 4B equilibrated with 10 mM MES buffer (pH = 5). 2 mL of the vesicle suspension was applied, and the separation was performed at a flow rate of 0.5 mL/min. Fractions of 1 mL were collected.
Absorption/turbidity measurements of each eluted fraction, performed with a spectrophotometer, indicate a good separation of the vesicles from the free enzyme. The optical density at 403 nm indicates two peaks, the first assigned to the turbid vesicles fraction, the second to free, non-entrapped HRPC with an absorption maximum at 403 nm due to the Soret band.

HRPC Activity Measurements
For measuring the activity of HRPC in solution, three stock solutions were freshly prepared: 1. An ABTS 2− stock solution was prepared by dissolving a few mg of ABTS 2− in 1 mL of 10 mM MES buffer solution (pH = 5). The exact concentration was calculated by UV absorption measurements (λ = 340 nm, ε340 = 36000 M −1 ·cm −1 ) [2]. The solution was kept in the dark at room temperature and used within 8 h. 2. A H2O2 stock solution (4 mM) was prepared by appropriate dilution with MilliQ water of a 30 wt% aqueous H2O2 solution. 3. An enzyme stock solution (4 mg/mL) was prepared by dissolving HRPC in 10 mM MES buffer (pH = 5). From this concentrated solution, a diluted enzyme stock solution (1 μM) was prepared by using the same buffer solution. Finally, for each measurement, two fresh stock solutions (100 nM and 2 nM) were prepared in plastic reaction tubes.
The activity measurements were carried out in the following way: MES Buffer solution (10 mM, pH = 5.0) was added to a polypropylene (PP) reaction tube to reach the assay volume of 1 mL, ABTS 2− (final concentration of 0.25 mM) and HRPC stock solution were mixed in the reaction tube. Immediately before the spectrophotometric analysis, H2O2 was added (final concentration of 80 μM). The mixture was transferred into a polystyrene (PS) cuvette (path length = 1 cm) and the change of the absorption spectrum of the reaction mixture was monitored as a function of time using a diode array spectrophotometer (Specord S600 from Analytik Jena). All measurements were repeated three times. The spectra were recorded at intervals of 10 sec immediately after the start of the reaction (up to 5 min).

Calibration Curves for HRPC Activity
Linear regression of the product absorbance at λmax = 414 nm as a function of time allowed an easy determination of enzymatic activity, read as the slope of the linear fit (dA414 / dt). Figure S5. HRPC concentration dependency of the absorbance of the reaction solution at 414 nm measured during the first 5 min of enzymatic reaction without addition of detergent to the substrate mixture (-■-) and with addition of Triton X-100 (0.1 %) (-•-). Each data point shown is the average from three measurements using the same stock solutions. The standard deviation is indicated with error bars. Deviation from linearity was evident for HRPC concentration higher than 350 pM HRPC.

Membrane Perturbation Studies
The snapshot from the MD trajectory at time 0 of the modified peptide azoALY shows the insertion of the peptide in membrane with a tilt angle of 48° and with the hydrophobic portion (Ala-Leu-Tyr-Leu-Ala) immersed in the core membrane. In the lest snapshot at a simulation time of 50 ns, the azoALY peptide is still anchored in the membrane, with the azo amino acid intercalate in the core membrane. Figure S6. Snapshot of (a) the starting configuration of the azoALY/POPC-POPG system; (b) the last frame at 50 ns of simulation time of the azoALY/POPC-POPG system; (c) the starting configuration of the ALY/POPC-POPG system; (d) he last frame at 50 ns of simulation time of the ALY/POPC-POPG system. The water molecules around the membrane are represented in stick style and salt in ball style. The membrane shape can be traced from the positions of phosphorus atoms, showed in red and the blue for the two membrane leaflets.
In Figure S7, the SCDs for POPC/POPG phospholipid chains around 5 Å from the peptides ALY (in red) and azoALY (in blue) are shown. In Figure S7a, the unsaturated chain is shown, and in Figure  S7b, the saturated one. Figure S7. Order parameter SCD for (a) the unsaturated oleic and (b) the saturated palmitoyl acyl chains of phospholipids in POPC/POPG/peptide (azoALY, blue curve; Aly red curve). Notes: On the Y-axis, the SCD is indicated; on the X-axis, the carbon atom position is reported, starting from the first (1) alpha carbon atom in the chains.