The palmitoyl groups of lung surfactant protein C reduce unfolding into a fibrillogenic intermediate

Lung surfactant protein C (SP-C) is a lipophilic peptide that converts from a monomeric α-helical state into β-sheet conformation and forms amyloid fibrils, a process which appears to be accelerated by removal of its two S-palmitoyl groups, and elevated amounts of non-palmitoylated SP-C are found in pulmonary alveolar proteinosis. Here, we used mass spectrometry to study the first step in fibrillogenesis of di-, mono- and non-palmitoylated SP-C. First, the individual decreases in concentration of monomeric α-helical forms of the three peptides in an acidified aqueous organic solvent mixture were monitored by electrospray (ES) mass spectrometry. Dipalmitoylated SP-C disappeared with a first-order rate constant of 0.01 h−1, corresponding to a t1/2 of 70 hours, while SP-C missing one or two palmitoyl groups disappeared with a rate constant of 0.02 h−1, t1/2 = 35 hours. This supports the suggestion that the acyl chains stabilise helical SP-C, and that small differences in helix stability can influence fibril formation. The rates of disappearance of the monomeric α-helical peptides are much faster than the disappearance of total soluble SP-C (t1/2 = 15 days for SP-C forms soluble after centrifugation at 20,000 g), which suggests that fibril formation is preceded by formation of soluble aggregates. Next, we used matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry to measure hydrogen → deuterium (H/2H) exchange in di-, mono- and non-palmitoylated SP-C in acidified aqueous organic solvents. All three species contain a rigid α-helix in their monomeric forms and no difference in deuterium uptake between SP-C with and without palmitoyl groups could be detected. The decreased stability of mono- and non-palmitoylated SP-C observed by ES mass spectrometry is thus not associated with partial unwinding of the helix in solution. Finally, SP-C was shown to unfold during the ES process (where ions are transferred from the solution to the gas phase) and the unfolded forms of di-, mono- and non-palmitoylated SP-C undergo H/2H exchange. This, together with the findings from MALDI H/2H experiments that the α-helix does not exchange, indicates that no partly helical intermediates exist and that the unfolding is highly cooperative.