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 alpha-helical state into beta-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 alpha-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 t(1/2) of 70 hours, while SP-C missing one or two palmitoyl groups disappeared with a rate constant of 0.02 h(-1), t(1/2)=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 alpha-helical peptides are much faster than the disappearance of total soluble SP-C (t(1/2)=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/(2)H) exchange in di-, mono- and non-palmitoylated SP-C in acidified aqueous organic solvents. All three species contain a rigid alpha-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/(2)H exchange. This, together with the findings from MALDI H/(2)H experiments that the alpha-helix does not exchange, indicates that no partly helical intermediates exist and that the unfolding is highly cooperative.