出版社：American Society for Biochemistry and Molecular Biology
摘要：The low density lipoprotein receptor (LDLR) is the prototypeof a family of cell surface receptors involved in a wide rangeof biological processes. A soluble low density lipoprotein receptor(sLDLR) and a tryptophan (Trp)-deficient variant human apolipoproteinE3 (apoE3) N-terminal domain (NT) were used in binding studies.The sole cysteine in apoE3-NT was covalently modified with anextrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine(AEDANS), and the protein was complexed with lipid. Incubationof sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine(DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancementin AEDANS fluorescence emission intensity (excitation, 280 nm)consistent with intermolecular energy transfer from excitedTrp in sLDLR to receptor-bound apoE. Ligand binding to sLDLRrequired calcium and was saturable. In competition binding assays,unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding tosLDLR more effectively than low density lipoprotein. Fluorescencechanges in this system reflected pH-dependent ligand bindingand release from sLDLR consistent with models derived from theX-ray crystal structure of the receptor at endosomal pH. Intermolecularenergy transfer from excited Trp in LDLR family members to fluorescentlytagged ligands represents a sensitive and convenient assay forthe characterization of the myriad molecular interactions ascribedto this family of receptor.Abbreviations: AEDANS, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine; apoE, apolipoprotein E; DMPC, dimyristoylphosphatidylcholine; EGF, epidermal growth factor; LDLR, low density lipoprotein receptor; sLDLR, soluble low density lipoprotein receptor; Trp, tryptophan
Supplementary key words fluorescence spectroscopy • apolipoprotein E • apolipoprotein B • assay • lipid • ligand binding
