Photocrosslinking and click chemistry enable the specific detection of proteins interacting with phospholipids at the membrane interface.

Abstract

New lipid analogs mimicking the abundant membrane phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine-alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively. Proteins crosslinked to the lipid analogs in inner mitochondrial membranes from Saccharomyces cerevisiae were detected and subsequently identified by mass spectrometry. Established interaction partners of phosphatidylcholine were found, as well as known integral and peripheral inner membrane proteins, and proteins that were not previously considered mitochondrial inner membrane proteins.

DOI: 10.1016/j.chembiol.2008.11.009

Cite this paper

@article{Gubbens2009PhotocrosslinkingAC, title={Photocrosslinking and click chemistry enable the specific detection of proteins interacting with phospholipids at the membrane interface.}, author={Jacob Gubbens and Eelco Ruijter and Laurence E V de Fays and J. Mirjam A. Damen and Ben de Kruijff and Monique Slijper and Dirk T. S. Rijkers and Rob M. J. Liskamp and Anton I.P.M. de Kroon}, journal={Chemistry & biology}, year={2009}, volume={16 1}, pages={3-14} }