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Journal of Lipid Research, Vol. 50, 1548-1555, August 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Semisynthesis and segmental isotope labeling of the apoE3 N-terminal domain using expressed protein ligation

Paul S. Hauser^*,, Vincent Raussens, Taichi Yamamoto^*, Gezman E. Abdullahi^**, Paul M. M. Weers^**, Brian D. Sykes and Robert O. Ryan^1,*,

^* Center for Prevention of Obesity, Diabetes, and Cardiovascular Disease, Children's Hospital Oakland Research Institute, Oakland, CA 94609
Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 94720-3104
Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Université Libre de Bruxelles, Brussels, Belgium
^** Department of Chemistry and Biochemistry, California State University, Long Beach, CA 90840
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

¹ To whom correspondence should be addressed. e-mail: rryan{at}chori.org

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that functions as a ligand for members of the LDL receptor family, promoting lipoprotein clearance from the circulation. Productive receptor binding requires that apoE adopt an LDL receptor-active conformation through lipid association, and studies have shown that the 22 kDa N-terminal (NT) domain (residues 1–183) of apoE is both necessary and sufficient for receptor interaction. Using intein-mediated expressed protein ligation (EPL), a semisynthetic apoE3 NT has been generated for use in structure-function studies designed to probe the nature of the lipid-associated conformation of the protein. Circular dichroism spectroscopy of EPL-generated apoE3 NT revealed a secondary structure content similar to wild-type apoE3 NT. Likewise, lipid and LDL receptor binding studies revealed that EPL-generated apoE3 NT is functional. Subsequently, EPL was used to construct an apoE3 NT enriched with ¹⁵N solely and specifically in residues 112–183. ¹H-¹⁵N heteronuclear single quantum correlation spectroscopy experiments revealed that the ligation product is correctly folded in solution, adopting a conformation similar to wild-type apoE3-NT. The results indicate that segmental isotope labeling can be used to define the lipid bound conformation of the receptor binding element of apoE as well as molecular details of its interaction with the LDL receptor.

Supplementary key words intein • apolipoprotein E • apolipoprotein

Abbreviations: AEDANS, N-(iodoacetyl)-N-(5-sulfo-1-naphthyl) ethylenediamine; apo, apolipoprotein; CD, circular dichroism; EPL, expressed protein ligation; HSQC, heteronuclear single quantum correlation; MESNA, 2-mercaptoethanesulfonic acid; NT, N-terminal; rHDL, reconstituted high density lipoprotein; sLDLR, soluble low density lipoprotein receptor; WT, wild-type

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