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.
Gustafsson
M
M
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, S-171 77, Sweden. magnus.gustafsson@mbb.ki.se
Griffiths
W J
WJ
Furusjö
E
E
Johansson
J
J
D017772:Amyloid Neuropathies / Q000378:metabolism; D000818:Animals; D011232:Chemical Precipitation; D006859:Hydrogen / Q000378:metabolism; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism; D010169:Palmitic Acids / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism*; D011663:Pulmonary Surfactants / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013552:Swine; D013816:Thermodynamics
D008744:Methylamines; D010169:Palmitic Acids; D011510:Proteolipids; D011663:Pulmonary Surfactants; D006859:Hydrogen; C023336:trimethylamine