New approaches for investigating membrane problems. A study by oriented circular dichroism and X-ray, neutron diffraction
Huang, Huey W.
Doctor of Philosophy
In this thesis I describe two frontier problems in membrane research. The first is about the outer membrane of gram-negative bacteria for which there was little prior research. Using lipopolysaccharide (LPS) bilayer as a model for the outer membrane, the problem of how antimicrobial peptides pass through the outer membrane were studied by oriented circular dichroism (OCD). The second is about non-lamellar structure of lipids that are important for membrane fusion. Two lipid structures, in the inverted HII phase and the distorted HII phase, respectively, are solved by a novel method through neutron scattering. Lipopolysaccharide bilayer was used to mimic the gram-negative bacterial outer membrane. X-ray diffraction patterns show that LPS forms a well aligned multilayer system. Some ions, such as barium (Ba++), were found to be located at the phosphate group in LPS headgroup. OCD spectra show that alpha-helical peptide mellitin and magainin have two different bound states to LPS multilayer that differs in the orientation with the respect to membrane. At high peptide concentration, they are in the active state, i.e. transmembrane state, whereas at low peptide concentration, they are in the inactive state in which they are oriented parallel to the membrane. In contrast, beta-sheet peptide protegrin only showed the transmembrane state. The structures of inverted HII phase of diphytanoylphosphatidylcholine (DPhPC) and a newly discovered distorted HII phase in dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylethanolamine (DOPE) mixture were studied by a new diffraction method to solve the phase problem in neutron scattering experiments. Through model construction, lipid packing patterns were studied. The DPhPC HII Hexgonal structure shows a circular headgroup packing, whereas the DOPC/DOPE mixture distorted HII phase shows that the. DOPC and DOPE are not uniformly distributed, possibly due to their different spontaneous curvatures. The structural information obtained in these studies will shed new light on the research on membrane fusion.