The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1

Abstract

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.

Real-time Measurement of Membrane Conformational States Induced by Antimicrobial Peptides: Balance Between Recovery and Lysis

“…1.25–40 μM peptide solutions were introduced consecutively to the same lipid bilayer to explore the effect of accumulative exposure of the bilayer surface to each peptide. The values of refractive index (n) and thickness (d) of an adsorbed isotropic single layer are determined by fitting measured phase changes in the transverse electric (T E ) and transverse magnetic (T M ) waveguide modes42629. The accumulative binding of Mag2 was first characterised by the TM and TE phase changes as a function of time as shown in Fig 2A and 2B.…”

“…The measured TM and TE phases were subsequently resolved into the mass of membrane-bound peptide and birefringence for both lipid bilayers42629. Plots of mass change versus time and birefringence versus time are shown in Fig.…”

“…While the mechanism of membrane permeabilization by Mag2 and its analogues has been studied on various model membrane systems11222324 and live cells25, there is limited information on the effect of Mag-A on bilayer structure. We have recently established the use of dual polarisation interferometry (DPI) to characterise the effect of peptide binding on the molecular ordering of the membrane through the measurement of birefringence426272829. We show here that the membrane bilayer undergoes a series of reversible structural changes upon Mag2 and Mag-A binding before a critical threshold concentration is reached which triggers a significant membrane disturbance.…”

α-Synuclein Senses Lipid Packing Defects and Induces Lateral Expansion of Lipids Leading to Membrane Remodeling

“…The mass per unit area of an adsorbed layer can be determined from thickness and refractive index using the Feijter formula, which uses the refractive index increments (dn/dc) of the lipid and protein, which are 0.135 and 0.182 ml/g, respectively, as reported previously (36,41). Alternatively, where the layer on the surface of the waveguide is birefringent, as is the case for SLBs, the refractive index (or thickness) of the layer can be assumed to be constant, and the thickness (or refractive index) and birefringence can be calculated (36,37). The birefringence or optical anisotropy measures the difference in refractive index of the two orthogonal polarizations caused by the net alignment of the lipid molecules perpendicular to the waveguide surface that creates an anisotropic system.…”

Interferometric Methods for Label-Free Molecular Interaction Studies

“…49,50 Farfield Sensors, Ltd. has been commercializing DPI-based biosensing systems since 2000 with the introduction of the AnaLight 250 26,51 and has most recently released the AnaLight 4D which is touted to enable the measurement of structural changes within lipid bilayers. 52,53 …”