Probing conformation of integral membrane protein phosphatidylserine synthase 2 by chemical crosslinking and mass spectrometry

Tuesday, October 09, 2012 — Poster Session I
1:00 p.m. – 3:00 p.m	Natcher Conference Center, Building 45	NIAAA	BIOCHEM-6

Authors

• A Kimura
• B Huang
• HY Kim

Abstract

We have previously demonstrated that docosahexaenoic acid-containing phospholipids are preferred substrates for biosynthesis of membrane phosphatidylserine catalyzed by phosphatidylserine synthase (PSS). While it is postulated that the conformational status of PSS is intimately coupled to its activity, the crystal structure of this integral membrane protein is not available at present. To provide molecular insight for the observed functional selectivity, we probed the conformation of PSS-2 using chemical crosslinking and mass spectrometry. Immunopurified PSS-2 was incubated with disuccinimidyl suberate (DSS), a lysine specific crosslinker. Monomeric or dimeric protein bands were separated by SDS-PAGE and subjected to tryptic digestion. Crosslinked peptides were analyzed by nanoLC-ESI-MS/MS. Three crosslinked lysine pairs, including K419-K425, K267-K284 and K265-K284, were identified in both monomeric and dimeric molecules, while the crosslinking between K252 and K351 was identified only in the dimeric PSS2 structure. As the crosslinked lysine pairs are located within 24 Å defined by DSS, the data provided for the first time the spatial distance information in the PSS2 structure. Additionally, the observed DSS-capping on K20, K133, K249, K252 and K263 suggested that these residues were easily accessible by the crosslinker. Monitoring the crosslinking profiles may give us insight into conformational changes of PSS2 accompanying its function.

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