Hydrogen/Deuterium Exchange and Aggregation of a Polyvaline and a Polyleucine α-Helix Investigated by Matrix-assisted Laser Desorption Ionization Mass Spectrometry

Abstract

The membrane-associated pulmonary surfactant protein C (SP-C), containing a polyvaline ␣-helix, and a synthetic SP-C analogue with a polyleucine helix (SP-C(Leu)) were studied by hydrogen/deuterium exchange matrix-assisted laser desorption ionization (MALDI) mass spectrometry. SP-C, but not SP-C(Leu), formed abundant amyloid fibrils under experimental conditions. In CD 3 OD/D 2 O, 91:9 (v/v), containing 2 mM ammonium acetate, SP-C(Leu) and SP-C exchanged 40% of their exchangeable hydrogens within 1 min. This corresponds to exchange of labile side-chain hydrogen atoms, hydrogens on the N-and C-terminal heteroatoms, and amide hydrogen atoms in the unstructured N-terminal regions. After ϳ300 h, four exchangeable hydrogen atoms in SP-C(Leu) and 10 in SP-C remained unexchanged. During this time period the ion current corresponding to singly charged SP-C decreased to <10% of the initial value due to the formation of insoluble aggregates that are not detected by MALDI mass spectrometry. In contrast, the ion current for SP-C(Leu) was maintained over this time period, although the peptides were incubated together. In combination, hydrogen/deuterium exchange and aggregation data indicate that the polyleucine peptide refolds into a helix after opening, while the unfolded polyvaline peptide forms insoluble ␤-sheet aggregates rather than refolding into a helix. The SP-C helix, but not the SP-C(Leu) helix, is thus in a metastable state, which may contribute to the recently observed tendency of SP-C and its precursor to misfold and aggregate in vivo.

Mutations linked to interstitial lung disease can abrogate anti-amyloid function of prosurfactant protein C

“…The efficiency of folding of transmembrane helices correlates with their helical propensity in aqueous solution; poly-Leu segments are folded into compact, possibly helical, structure already in the translocon while poly-Val has a more extended conformation [42]. SP-C is highly inefficient in forming helical structure, in contrast to polyVal→polyLeu substituted variants [8,30,33,34]. It is therefore likely that formation of the proSP-C polyVal transmembrane helix is comparatively inefficient [24].…”

“…MALDI mass spectrometry confirmed the presence of SP-C in the aggregates (data not shown). In the presence of CTC at a molar ratio of about 0.5-1 versus SP-C, SP-C fibril formation was fully prevented after 7 days ( surfactant preparation containing phospholipids and an SP-C analogue with a polyLeu stretch replacing the polyVal part [33], was incubated in the same manner as described above. In this case no amyloid fibrils were observed by electron microscopy (Fig.…”

Hydrogen exchange mass spectrometry for the analysis of protein dynamics

“…She has used MALDI HX MS to identify several binding sites in the cyclic-AMP-dependent protein kinase (PKA) complex with the kinase inhibitor and ATP (Mandell, Falick, & Komives, 1998a) and to determine the antibody-antigen recognition site for a monoclonal antibody raised against human thrombin (Baerga-Ortiz et al, 2002). Others have reported on the use of MALDI to probe the folding and assembly of viral capsids (Tuma et al, 2001) and to investigate protein conformational changes as a result of polyvaline and polyleucine a-helix aggregation (Hosia, Johansson, & Griffiths, 2002). Topological information about yeast F1-ATPase, a supramolecular hetero-oligomeric protein complex of 370 kDa, was obtained using MALDI as the ionization method (Nazabal et al, 2003).…”

Proteolytic generation and aggregation of peptides from transmembrane regions: lung surfactant protein C and amyloid ?-peptide

“…It is likely that removal of SP-C from the membrane promotes aggregation, but high concentrations of SP-C in solution can also form β-sheet aggregates in the presence of membrane lipids [59]. The irreversible nature of the first step is deduced from the lack of detectable hydrogen/deuterium exchange in the central part of the SP-C helix, as observed by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry [55,60]. The proposal that unfolding of the helix precedes aggregation is in line with the observed first-order kinetics of disappearance of helical SP-C.…”