Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and antibacterial agent
Fig 13
Surface plasmon resonance (SPR) analysis of temporin binding to negatively charged DMPC/DMPG 3:1 (mol:mol) LUVs.
A, binding of temporins directly to the L1 sensor chip surface. SHa and [K3]SHa injected at a concentration of 5 μM (20 μl during 1 min) interact with the carboxymethylated dextran containing covalently attached alkyl chains, as indicated by the significant amount of temporin non-specific binding (SHa: 197 RU, [K3]SHa: 240 RU) remaining on the sensor chip surface after the end of peptide injection. B and C, binding of SHa (B) and [K3]SHa (C) after injection of BSA. In contrast, no peptide interaction was observed after binding of 0.2 mg/ml BSA (15 μl injected during 3 min) to the sensor chip surface followed by injection of SHa or [K3]SHa (5 μM). D, complete SPR cycle used for the binding of temporins. In the example, 0.2 mg/ml BSA was first injected (15 μl during 3 min) on the L1 surface to prevent non-specific binding of temporins and was followed by an injection (2 μl during 2 min) of 0.2 mg/ml DMPC/DMPG LUVs and then of peptide (300 nM of SHa in the example; 20 μl during 1 min). Complete regeneration of the surface was obtained using 40 mM of the detergent n-octyl-β-D-glucopyranoside (OG) (30 μl injected during 1 min). E and F, determination of the binding affinity of temporins SHa (E) and [K3]SHa (F). Peptides diluted in HBS-N buffer were tested at different concentrations (0 to 300 nM) for their binding to DMPC/DMPG LUVs. The baseline corresponds to HBS-N alone. The following KD values were calculated by BIAevaluation software analysis: KD (SHa) = 1.3 ± 0.4 x 10−7 M, χ2 = 3.7 ± 1.3 (n = 3); KD ([K3]SHa) = 3.1 ± 0.7 x 10−8 M, χ2 = 3.2 ± 0.7 (n = 3). Chi2 (χ2) values below 10 indicate a good fit of the Langmuir (1:1) binding model. G and H, selective SPR binding of temporins SHa (G) and [K3]SHa (H) toward anionic model membranes. Negatively charged DMPG or zwitterionic DMPC LUVs were injected onto the L1 sensor chip precoated with BSA (0.2 mg/ml). Temporins (500 nM) were then injected, and binding to the DMPG (solid line) and DMPC (dashed line) LUVs was monitored. RU: resonance units; SI: start of injection; EI: end of injection. The curves correspond to a single experiment representative of three different experiments.