期刊
ACS CHEMICAL BIOLOGY
卷 16, 期 8, 页码 1493-1507出版社
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.1c00364
关键词
-
资金
- National Institutes of Health [R01 MH110550, R01 HL067773, R01 GM108799, T32 HL134635, F31 HL142167]
- Taylor Family Institute for Innovative Psychiatric Research
Cholesterol analogues containing aliphatic diazirine or TPD groups were used as photoaffinity labeling reagents to study cholesterol binding sites on GLIC. The probes showed differences in photolabeling efficiencies and residues identified, indicating complementary information provided by the two classes of probes. Photoaffinity labeling using these reagents in mammalian cell membranes revealed differences in the pattern of labeled proteins, highlighting their utility in future studies of cholesterol biology.
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.
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