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Originally published In Press as doi:10.1194/jlr.D600001-JLR200 on February 8, 2006

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Journal of Lipid Research, Vol. 47, 1091-1096, May 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Methods

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

Taichi Yamamoto, Johanne Lamoureux and Robert O. Ryan1

Lipid Biology in Health and Disease Research Group, Children's Hospital Oakland Research Institute, Oakland, CA 94609

Published, JLR Papers in Press, February 8, 2006.

1 To whom correspondence should be addressed. e-mail: 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 low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was 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 intermolecular 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. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to 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


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V. Gupta, V. Narayanaswami, M. S. Budamagunta, T. Yamamato, J. C. Voss, and R. O. Ryan
Lipid-induced Extension of Apolipoprotein E Helix 4 Correlates with Low Density Lipoprotein Receptor Binding Ability
J. Biol. Chem., December 22, 2006; 281(51): 39294 - 39299.
[Abstract] [Full Text] [PDF]


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Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.