NMR Structures of the C-Terminal Segment of Surfactant Protein B in Detergent Micelles and Hexafluoro-2-propanol

Abstract

Although the membrane-associated surfactant protein B (SP-B) is an essential component of lung surfactant, which is itself essential for life, the molecular basis for its activity is not understood. SP-B's biophysical functions can be partially mimicked by subfragments of the protein, including the C-terminus. We have used NMR to determine the structure of a C-terminal fragment of human SP-B that includes residues 63-78. Structure determination was performed both in the fluorinated alcohol hexafluoro-2-propanol (HFIP) and in sodium dodecyl sulfate (SDS) micelles. In both solvents, residues 68-78 take on an amphipathic helical structure, in agreement with predictions made by comparison to homologous saposin family proteins. In HFIP, the five N-terminal residues of the peptide are largely unstructured, while in SDS micelles, these residues take on a well-defined compact conformation. Differences in helical residue side chain positioning between the two solvents were also found, with better agreement between the structures for the hydrophobic face than the hydrophilic face. A paramagnetic probe was used to investigate the position of the peptide within the SDS micelles and indicated that the peptide is located at the water interface with the hydrophobic face of the helix oriented inward, the hydrophilic face of the helix oriented outward, and the N-terminal residues even farther from the micelle center than those on the hydrophilic face of the R-helix. Interactions of basic residues of SP-B with anionic lipid headgroups are known to have an impact on function, and these studies demonstrate structural ramifications of such interactions via the differences observed between the peptide structures determined in HFIP and SDS.

Design of Surfactant Protein B Peptide Mimics Based on the Saposin Fold for Synthetic Lung Surfactants

“…Similar to the N-terminal domain, the positively charged helical C-terminus of SP-B may be involved in lung functions, as synthetic peptides either directly (e.g., SP-B residues 63–78: GRMLPQLVCRLVLRCS) (PDB: 1RG3, 1RG4) or indirectly [e.g., KL4 (21 residues): KLLLLKLLLLKLLLLKLLLLK] representing the C-terminus may adopt helical conformations [22,65–67] and promote in vitro [65,66,68,69] and in vivo [68,70,71] surfactant activities mimicking those of the native protein. Importantly, lucinactant, a new total synthetic lipid-peptide preparation that includes the KL4 peptide designed to loosely mimic the C-terminal domain of SP-B, has been tested in two clinical trials with preterm infants at risk for RDS [72,73].…”

Synthetic Pulmonary Surfactant Preparations: New Developments and Future Trends

“…The structure of some of these proteins has, in most cases, been solved in a membrane-free state. However, a molecular structure of SP-B, the only member of the saposin family that is permanently associated with membranes, is still not available despite the fact that some molecular modelling efforts have been reported [69,70]. Numerous studies have approached the analysis of structural determinants in SP-B by means of indirect techniques such as circular dichroism [71,72] [73], fluorescence [74] or infrared spectroscopies [75,76], or electron spin resonance (ESR) [77].…”

Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques

“…TFE and HFIP media have been used in the past to determine the structure of similar transmembrane proteins and receptor fragments because of their ability to stabilize their alpha-helical structure [14,28,39,40,41]. The secondary structures stabilized by either TFE and HFIP were often assumed to reflect conformations that prevail in the early stages of protein folding [42,43].…”