Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents

Kathiresan Krishnan, Mingxing Qian, McKenna Feltes, Zi Wei Chen, Sarah Gale, Lei Wang, Yusuke Sugasawa, David E. Reichert, Jean E. Schaffer, Daniel S. Ory, Alex S. Evers, Douglas F. Covey

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

Original languageEnglish
Pages (from-to)1493-1507
Number of pages15
JournalACS Chemical Biology
Volume16
Issue number8
DOIs
StatePublished - Aug 20 2021

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