NMR structural studies of lung surfactant protein B (SP-B) peptides

Mammalian lungs are composed of millions of tiny air sacs called alveoli where gas exchange takes place. The inner surface of alveoli is coated by an aqueous layer to prevent it from drying up. However, because the attraction between water molecules is stronger than the force between water and air, a surface tension is created at the air-water interface. The tension tends to collapse the alveoli and increase the work of breathing. Lung surfactant is a material that counteracts these tendencies by reducing the surface tension to extremely low values and thus prevents the alveolar collapse and also eases the work of breathing. The lung surfactant material is a mixture of lipids and proteins. Surfactant Protein B (SP-B) is an essential component of the surfactant and is thought to function by facilitating large-scale rearrangement of the lipid molecules and stabilizing the structures. However, neither the structural basis for this ability nor the physiological ramifications of lipid rearrangements are yet understood. SP-B is a lipid-associated hydrophobic protein, which makes it difficult to address with X-ray or conventional solution NMR structural techniques. These difficulties have been addressed in NMR structural studies by the use of fluorinated organic solvents and lipid micelles to solubilize the proteins. The present work has focused on SP-BcTERM and Mini-B, two peptide fragments of SP-B that retain significant biological activity of the full-length protein. The structural features of these peptides were studied using high-resolution solution NMR. Firstly, the conformational features of SP-BcTERM and its interactions with lipids were investigated in micelles mimicking the lipid environment in the lungs. The peptide exhibited the ability to cause aggregation of micelles formed from lipids similar to those found in lungs. This was indicative of the large-scale lipid rearrangement and stabilization of structures facilitated by SP-B in natural surfactant. In the second and main phase of current research, the structure of Mini-B was determined in the structureinducing fluorinated organic solvent hexafluoroisopropanol (HFIP). The peptide was found to consist of two a-helices at the termini connected by an unstructured loop at the middle. These studies help to define the structural properties that underlie SP-B's function and provide a platform to probe the lipid-protein interactions that are responsible for the ability of lung surfactant to dramatically lower the surface tension at the air-water interface in alveoli

The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation

AbstractThe reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell–cell, rather than virus–cell, membrane fusion. The 36–40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell–cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome–liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol

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

Bicelle composition-dependent modulation of phospholipid dynamics by apelin peptides.

Apelin peptides are cognate ligands for the apelin receptor, a G-protein coupled receptor (GPCR). The apelinergic system plays critical roles in wide-ranging physiological activities including function and development of the central nervous and cardiovascular systems. Apelin is found in 13-55 residue isoforms in vivo, all of which share the C-terminal portion of the preproapelin precursor. Characterization of high-resolution structures and detergent micelle interactions of apelin-17 led to a two-step membrane-catalyzed binding and GPCR activation mechanism hypothesis recapitulated in longer isoforms. Here, we examine interactions of the apelin-13 and -17 isoforms with isotropic zwitterionic and mixed zwitterionic-anionic lipid bicelles to test for hallmarks of membrane catalysis in a more physiological membrane-mimetic environment than a micelle. Specifically,The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Lung Surfactant Protein A (SP-A) Interactions with Model Lung Surfactant Lipids and an SP-B Fragment

Surfactant protein A (SP-A) is the most abundant protein component of lung surfactant, a complex mixture of proteins and lipids. SP-A performs host defense activities and modulates the biophysical properties of surfactant in concerted action with surfactant protein B (SP-B). Current models of lung surfactant mechanism generally assume SP-A functions in its octadecameric form. However, one of the findings of this study is that when SP-A is bound to detergent and lipid micelles that mimic lung surfactant phospholipids, it exists predominantly as smaller oligomers, in sharp contrast to the much larger forms observed when alone in water. These investigations were carried out in sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), lysomyristoylphosphatidylcholine (LMPC), lysomyristoylphosphatidylglycerol (LMPG), and mixed LMPC + LMPG micelles, using solution and diffusion nuclear magnetic resonance (NMR) spectroscopy. We have also probed SP-A’s interaction with Mini-B, a biologically active synthetic fragment of SP-B, in the presence of micelles. Despite variations in Mini-B’s own interactions with micelles of different compositions, SP-A is found to interact with Mini-B in all micelle systems and perhaps to undergo a further structural rearrangement upon interacting with Mini-B. The degree of SP-A–Mini-B interaction appears to be dependent on the type of lipid headgroup and is likely mediated through the micelles, rather than direct binding