Structures and interactions of lung surfactant protein B (SP-B) peptides

Lung surfactant is a complex mixture of lipids and proteins that enables normal breathing by reducing the surface tension at the alveolar air-water interface, and additionally provides the first line of defense against inhaled microbes in the lungs. Surfactant protein B (SP-B) is an indispensable component of lung surfactant system and absolutely essential for the survival of mammals. SP-B is thought to function by facilitating large-scale rearrangements of lipid structures at various stages of the breathing cycle. However, neither the structural mechanisms for this ability nor the physiological ramifications of the lipid rearrangements are yet understood, in part because a high-resolution structure of SP-B has not been determined. As is generally the case for membrane and other lipid-associated hydrophobic proteins, the production of an SP-B sample for structural studies has been very challenging and unsuccessful to date. Interestingly, synthetic fragments of SP-B retain substantial biological activity when compared to the full-length protein. This Ph.D. research has applied solution nuclear magnetic resonance (NMR) methods to three SP-B-based peptides to reveal at least some of the critical structural features and lipid/protein interactions that presumably underlie the functional mechanisms of SP-B in physiological conditions. -- The high-resolution structure of Mini-B, an N-terminal - C-terminal construct of SP-B that exhibits even better performance than the full-length protein in rat models, is determined in the presence of surfactant lipid-mitnetic sodiumdodecylsulfate (SDS) micelles. Mini-B consists of two a-helices with a projecting tryptophan anchor and displays a strikingly amphipathic surface. The structure of Mini-B appears very well-suited for making strong interactions with surfactant phospholipid analogues. Indeed, Mini-B binds both anionic and zwitterionic micelles composed of SDS, dodecylphosphocholine (DPC), lysomyristoylphosphatidylcholine (LMPC), lysomyristoylphosphatidylglycerol (LMPG) and mixed LMPC/LMPG, and induces substantial rearrangements of the micelle structures. To prepare a foundation for directly probing the interaction between Mini-B and surfactant protein A (SP-A), the conformation and lipid interactions of SP-A are investigated separately in the presence of micelles composed from the same model surfactant lipids. SP-A also binds both zwitterionic and anionic micelles. Surprisingly, in the presence of micelles, SP-A exists predominantly as smaller oligomers, in sharp contrast to the octadecamers observed when in an aqueous environment, and the form in which SP-A has long been presumed to function. Mini-B appears to interact with SP-A in all micelle systems, although the interaction is indirect and the degree of the interaction is dependent on the type of detergent/lipid headgroup. Next investigated are the changes to SP-B's structure and lipid interactions brought about by tryptophan oxidation, a modification which is thought to be a major contributor to acute respiratory distress syndrome (ARDS). Replacement of tryptophan by one of its oxidized forms, kynurenine, substantially disrupts the helical structure of SP-B8-25, an N-terminal fragment of SP-B, and also affects its interactions with the micelles. Lastly, as a step towards the determination of the structure of full-length SP-B, the overall conformation of Maxi-Bcr, the C-terminal half of SP-B, has been investigated in the organic solvent hexafluoroisopropanol (HFIP) and SDS micelles. -- SP-B is indispensable for life, but the molecular basis for its activity is not yet understood. The findings of this Ph.D. research contribute to the ongoing endeavor in characterizing SP-B's structure-function relationships and its mechanisms of lipid/protein interactions that are crucial for lung surfactant function. This work also provides a strong foundation for future studies on the conformation and interactions of full-length SP-B