Characterization of low-density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

Taichi Yamamoto, Johanne Lamoureux, and Robert O. Ryan

Children's Hospital Oakland Research Institute, Oakland, CA 94609

Corresponding Author: rryan{at}chori.org

The low-density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble fragment of human LDLR (sLDLR) and a tryptophan-deficient variant human apolipoprotein (apo) E3 N-terminal (NT) domain were employed in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe (AEDANS) and the protein complexed with lipid. Incubation of sLDLR with AEDANS-Trp null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation 280 nm) consistent with inter-molecular energy transfer from excited Trp in sLDLR to receptor bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low-density lipoprotein. Fluorescence changes in this system reflected pH dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Inter-molecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for characterization of the myriad molecular interactions ascribed to this family of receptor.

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