Investigation of the Artificial Antimicrobial Peptides GW-H1 and GW-Q4 on the Mechanism of Membrane Permeation with Oriented Circular Dichroism (OCD), Lamellar X-ray Diffraction (LXD) and Small Angle X-ray Scattering (SAXS)
Date Issued
2010
Date
2010
Author(s)
Jian, Jia-Nan
Abstract
Antimicrobial peptides (AMPs) play important roles in the host innate defense mechanism in many plants, insects, and mammals. It is believed that AMPs may interact with the microbial membranes and kill the target cells. On the other hands, the extensive use of classical antibiotics has led to the growing emergence of many resistant strains of pathogenic microorganisms in recent years. Therefore, the development of novel therapeutic agents that could overcome the antimicrobial resistance has become a very critical issue.
The mechanism for the aforementioned antimicrobial activity has been considered as the membrane-peptide interactions and the subsequent pore- forming that lead to the permeation of biomembranes. Several models have been proposed according to the membrane structure types during pore-formation: “barrel-stave”, “carpet” and “toroidal-pore”. Based on the previous study of our collaborators, a series of cationic α-helical peptides with 20 amide acids has been designed and synthesized according to four structural determinants: charge, polar angle, hydrophobicity, and hydrophobic moment. Two of such de novo designed AMPs, GW-H1 (Q＝＋4, θ＝140°, H＝-0.115, MH＝0.344) and GW-Q4 (Q＝＋4, θ＝140°, H＝-0.043, MH＝0.344), exhibited the most significant antimicrobial activity and selectivity against various Gram-positive and Gram-negative bacteria, including several vibrio strains. Results form the related calcein leakage experiments and circular dichroism spectra are used to infer that the antimicrobial activity of GW-H1 and GW-Q4 should rely on the direct interaction with prokaryotic membranes and the concomitant penetration effect that can lead to the microbial death.
In this study, to distinguish the type of membrane-peptide interactions, which will allow us to deduce the properties of such interaction in detail, and to understand the difference in mechanism between artificial and natural AMPs, we apply DOPC/DOPG (3:1) membranes as a bacterial cell membrane system to investigate the physical factors participating in the interaction. Peptides adopted are GW-H1 and GW-Q4 (artificial); melittin and pleurocidin (natural). Both the lamellae and liposomes were used as (apparatus) platforms for membrane. The biophysical techniques applied include the followings. (i) Oriented circular dichroism (OCD). This is used to monitor the peptide orientation: parallel means surface adsorbed, whereas perpendicular means pore wall attached or membrane integrated. (ii) Lamellar X-ray diffraction (LXD): used to measure the change in thickness of membrane bilayer in solid state. (iii) Small angle X-ray scattering (SAXS): used to measure the change in thickness of membrane bilayer of small unilamellar vesicles (SUVs) in solution. The physical measurements are conducted during experiments via observing the peptide orientations, the change in membrane thickness and the change in size of liposomes, for which all as an individual function of peptide-to-lipid molar ratio (P/L).
The results show that artificial antimicrobial peptide GW-H1 and GW-Q4 behave in a different manner from the natural antimicrobial peptides melittin and pleurocidin. It is indicated that GW-H1 and GW-Q4 adsorbed onto the biomembrane surface continuously and in parallel, instead of attaching perpendicularly in membrane per se. Therefore the membrane becomes thinner and thinner, even without digging a pore. This coincides with the data on particle size measurement from DLS (Dynamic Light Scattering), suggesting the liposome membrane structure has not been seriously interrupted, damaged or deformed. However, the calcein leakage experiments strongly suggested the exchange of materials through membrane. How can we explain this? According to the literatures, noting that the physical properties of lipid membranes, as membranes lipids are getting closer to each other, they will be influenced by the thermal fluctuation force and moving aparts, a condition that may cause the transient pores to occur on the membrane surfaces. We speculate that the synthetic antibacterial peptide GW-H1 and GW-Q4 should be accumulating on the membranes and force the membrane structure to become more fragile, as the surface tension will be increased during the membranes are thinnened. This will probably cause the transient pores to occur by a higher frequency or to a larger extent in size, which is in a similar way as being influenced by thermal fluctuations. This condition may result in the temporary loss of barrier functions of the biomembrane.
In contrast, the natural peptide melittin apparently inserts itself into the membrane as described for the toroidal-pore model. Our results provide clear evidence for such model and working hypothesis, according to the observation for peptide distributions both in parallel and in perpendicular, as well as the change in membrane thickness from both SAXS and LXD data. Besides, the critical concentration for pore-formation (P/L)* of melittin in DOPC/DOPG (3:1) is ~1/200, which is only half value compared with that in pure DOPC (~1/99). This finding is in line with the previous concept that the initial steps of cationic AMPs when binding onto the microbial membrane surface are essentially relying on the electrostatic interactions. In our case, the addition of DOPG into DOPC mixture indeed brings more negative charges to a neutral system. The effect of pleurocidin on membrane is similar to melittin. The pleurocidin may cause the penetration on the microbial membrane by forming pores in toroidal-pore model. This is consistent with the comprehension from electrophysiological data described in literature. Moreover, our preliminary data show that pleurocidin may interact with membranes in an even stronger manner, for which severe disruption of membrane vesicles were observed during experiments, and unexpected huge error bars had to be taken care of.
In the present study, we applied the most advanced synchrotron technology to a local tradition of research, which has been since long excellent but then difficult to explore more in a decent way. That is, an extensive scientific focus on the interactions between toxins/peptides and biomembranes. Biophysical measurements of objective parameters enable the detailed study for the aforementioned topic to be approached in a rigid way. Deduction of the high resolution data from operation of synchrotron light sources indeed sheds lights into this new platform of research. Our studies may even provide a standard procedure in methods and methodology for related membrane-peptide research. However, it is still difficult and complicated to comprehend the derivations directly from the structural determinants in designing artificial peptides to the mechanism categorization. This will rely on further approaches in the near future.
Subjects
antimicrobial peptide
oriented circular dichroism
lamellar x-ray diffraction
small angle x-ray scattering
dynamic light scattering
liposome
thermal fluctuation
transient pore
Type
thesis
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