Cooperative phenomena of antimicrobial peptides in membranes: A study by neutron and X-ray diffraction

Abstract

Gene-encoded membrane-active antimicrobial peptides permeablize bacterial plasma membranes without harming the host cells. Furthermore, although most peptides exhibit a broad spectrum of activity against microbes, different peptides preferentially kill different pathogens. Understanding such cell-type specificity is not only fundamental to cell biology but also crucial to potential pharmaceutically applications of antimicrobial peptides. Accumulated evidence indicates that the antimcrobial peptides target the lipid matrix of the plasma membranes. Therefore we focus on the physical states of the peptides bound to lipid bilayers. This thesis describes studies of lipid-peptide systems in the form of aligned multi-lamellae with new neutron and X-ray diffraction techniques developed specifically for such systems, under various conditions with improved temperature and relative humidity control. These technique allow the most detailed structural investigation on the supramolecular assemblies formed by these peptides in model lipid membranes. Interesting phenomena were observed. Peptides form transmembrane pores in fluid lipid bilayers. The sizes of various peptide pores were determined by fitting neutron scattering data with the theory of scattering. By manipulating the temperature and the hydration level of the samples, we observed position correlations developed between the pores located in neighboring bilayers that eventually became long-range and the transmembrane pores were crystallized in lipid membranes for the first time. Diffraction data of the crystallized pores were measured with synchrotron radiation using samples on ultra-thin Si3N4 substrate for transmission X-ray diffraction. A number of different crystalline phases were found. One example is the ABC stacking hexagonal structure, surprisingly also found in pure diphytanoyl phosphatidylcholine samples. Correlating the diffraction data with circular dichroism and other experimental evidence, we separate the pore structures into two categories described by the barrel-stave model and the toroidal model. The implication of these results on the peptide's cell-type specificity is also discussed in terms of the properties of the lipids and environmental variables.