Chemistry & Biology
Volume 16, Issue 1, 30 January 2009, Pages 3-14
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Article
Photocrosslinking and Click Chemistry Enable the Specific Detection of Proteins Interacting with Phospholipids at the Membrane Interface

https://doi.org/10.1016/j.chembiol.2008.11.009Get rights and content
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Summary

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.

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Cited by (0)

4

Present address: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239, USA

5

Present address: Department of Organic and Inorganic Chemistry, VU University, Amsterdam, The Netherlands

6

Present address: Federal Agency for Drugs and Health Products, Bruxelles, Belgium

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Present address: Membrane Enzymology, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands