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 a myloid 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 t o study the first step in fibrillogenesis of di-, mono- and non-palmitoylat ed SP-C. First, the individual decreases in concentration of monomeric alph a -helical forms of the three peptides in an acidified aqueous organic solv ent mixture were monitored by electrospray (ES) mass spectrometry. Dipalmit oylated SP-C disappeared with a first-order rate constant of 0.01 h(-1), co rresponding to a t(1/2) of 70 hours, while SP-C missing one or two palmitoy l 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, a nd that small differences in helix stability can influence fibril formation . The rates of disappearance of the monomeric alpha -helical peptides are m uch faster than the disappearance of total soluble SP-C (t(1/2) = 15 days f or SP-C forms soluble after centrifugation at 20,000 g), which suggests tha t 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/H-2) exchange in di-, mono- and non-p almitoylated 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 de tected. The decreased stability of mono- and non-palmitoylated SP-C observe d 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 proc ess (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/H-2 exch ange. This, together with the findings from MALDI H/H-2 experiments that th e alpha -helix does not exchange, indicates that no partly helical intermed iates exist and that the unfolding is highly cooperative. (C) 2001 Academic Press.