Experimental and molecular dynamics study of the interactions of lipid membranes and the pulmonary surfactant-associated protein B in model pulmonary surfactant systems

• Author / Creator

  Wu,Min

• The pulmonary surfactant exists as an active film, easing the work of lung by fast absorption and desorption during respiratory cycles, which the principle function of both the lipid and protein components is to reduce the surface tension of the air-liquid interface. The pulmonary surfactant is not simply a mixture of lipids and proteins, but a high ordered architecture, where the pulmonary proteins play crucial roles in structure establishment. Of all the pulmonary surfactant proteins, SP-A, SP-B, SP-C and SP-D, the pulmonary surfactant associated-protein B (SP-B) is critically required for the respiratory process. The mechanisms of the SP-B interaction with lipids involved in the lipid layers of surfactant film remains unsettled, from details about the effects of protein in lipid monolayer to protein-mediated membrane fusion when the multilayer structure of stored surfactant sites, e.g. lamellar bodies, were unpacked into the active film. One of the difficulties in understanding the protein-lipid interactions lies in the complexity of the structural conversion of SP-B and lipid membranes. Molecular dynamics simulation is a powerful tool for probing the free energy profiles between membranes as well as structure and dynamics variations of biological molecules in the membrane system, and surface force apparatus provides a high-resolution way to directly measure the molecular interactions. We experimentally prepared Langmuir Blodgett monolayers after different compression-expansion cycles and simulated the lipid monolayer in the presence of SP-B to study the role of protein in the interfacial properties of the dipalmitoylphosphatidylcholine (DPPC) monolayer. Using atomic force microscopy and transmission electronic microscopy imaging, we found that SP-B containing DPPC monolayers generated a network with a highly detailed structure, accompanying an enhanced re-spreading efficiency characterized by fewer aggregates observed following the monolayer expansion from high surface pressure. Molecular dynamics studies indicated that SP-B induced a local groom by disordering the packing of lipid chains upon monolayer compression. SP-B might provide nucleation sites during monolayer compression, which possibly explained how the network of nano-domains was developed and agreed the morphological observation from the experiment. The interactions between lipid layers determined the initialization of transformation from intermediate multilayered state of pulmonary surfactant to the final active film. For probing the model lipid bilayer-bilayer interactions, we used umbrella sampling molecular dynamics simulations to characterize the energy minimum for the opposing DPPC bilayers. The simulated energy minimum between lipid bilayers was highly matched with the experimental results. When the two bilayers were compressed together, the lipid chains were found interdigitated due to the high pressure and dehydration of lipid head groups. The structure and dynamics of water molecules showed the confinement of water molecules with the average separation of bilayers reduced to ~ 0.6 nm. The release of lamellar bodies initiates with the fusing of their limiting membrane with the type II pneumocytes membrane. To further investigate the function of SP-B involved in membrane fusing, we directly measured molecular forces between model bilayers. As confirmed by the thickness variation measured from surface force apparatus, fusing was observed for proteins containing DPPC bilayers. Large adhesive energy was obtained and expected to mainly originate from hydrophobic interactions between ɑ-helix and lipid chains. For palmitoyloleoylphosphoglycerol (POPG) involved interactions between membranes, SP-B modified the fusing process of bilayers with obvious hysteresis for the approach and separation pathways, indicating the protein-mediated lipid bilayer reconstruction. These results shed light on the information about the structural conversion of SP-B and membranes (monolayer and bilayer) involved in surfactant film systems and illustrate a general method of combined experiments and simulations for studying the lipid-protein and lipid-lipid interactions at a molecular level, with applications in seeking a plausible surfactant candidate for surfactant replacement therapy.

• Subjects / Keywords

  • Surfactants
  • Protein B
  • Lipid membranes

• Graduation date

  Spring 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3W95136K

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.