Quenching of tryptophan fluorescence in a highly scattering solution: Insights on protein localization in a lung surfactant formulation

PLoS One. 2018 Aug 3;13(8):e0201926. doi: 10.1371/journal.pone.0201926. eCollection 2018.

Abstract

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic surfactant developed for respiratory distress syndrome replacement therapy in pre-term newborn infants. CHF5633 contains two phospholipids (dipalmitoylphosphatidylcholine and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol sodium salt), and peptide analogues of surfactant protein C (SP-C analogue) and surfactant protein B (SP-B analogue). Both proteins are fundamental for an optimal surfactant activity in vivo and SP-B genetic deficiency causes lethal respiratory failure after birth. Fluorescence emission of the only tryptophan residue present in SP-B analogue (SP-C analogue has none) could in principle be exploited to probe SP-B analogue conformation, localization and interaction with other components of the pharmaceutical formulation. However, the high light scattering activity of the multi-lamellar vesicles suspension characterizing the pharmaceutical surfactant formulation represents a challenge for such studies. We show here that quenching of tryptophan fluorescence and Singular Value Decomposition analysis can be used to accurately calculate and subtract background scattering. The results indicate, with respect to Trp microenvironment, a conformationally homogeneous population of SP-B. Trp is highly accessible to the water phase, suggesting a surficial localization on the membrane of phospholipid vesicles, similarly to what observed for full length SP-B in natural lung surfactant, and supporting an analogous role in protein anchoring to the lipid phase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Fluorescence
  • Humans
  • Lipid Bilayers / chemistry
  • Models, Theoretical
  • Peptide Fragments / chemistry
  • Phosphatidylcholines / chemistry
  • Pulmonary Surfactant-Associated Protein B / chemistry
  • Pulmonary Surfactant-Associated Protein C / chemistry
  • Pulmonary Surfactants / chemical synthesis
  • Pulmonary Surfactants / chemistry*
  • Tryptophan / chemistry*

Substances

  • CHF5633
  • Lipid Bilayers
  • Peptide Fragments
  • Phosphatidylcholines
  • Pulmonary Surfactant-Associated Protein B
  • Pulmonary Surfactant-Associated Protein C
  • Pulmonary Surfactants
  • Tryptophan

Grants and funding

Chiesi Farmaceutici has funded this study providing financial support in the form of Chiesi employees salaries [BP, FS and SC] and research materials to Prof. Stefano Bettati but did not have any additional role in the study design, data collection and analysis, decision to publish and preparation of the manuscript. The specific roles of Chiesi employee authors are articulated in the “authors contributions” section.