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Molecular & Cellular Proteomics 1:592-597, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

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

Waltteri Hosia, Jan Johansson and William J. Griffiths

From the Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

To whom correspondence should be addressed. Tel.: 46-8-7287721; Fax:46-8-339004; E-mail: william.griffiths{at}mbb.ki.se

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 CD₃OD/D₂O, 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 300 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 ß-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.

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