Semisynthesis and segmental isotope labeling of the apoE3 N-terminal domain using expressed protein ligation.
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.
Hauser
Paul S
PS
Center for Prevention of Obesity, Diabetes, and Cardiovascular Disease, Children's Hospital Oakland Research Institute, Oakland, CA 94609, USA.
Raussens
Vincent
V
Yamamoto
Taichi
T
Abdullahi
Gezman E
GE
Weers
Paul M M
PM
Sykes
Brian D
BD
Ryan
Robert O
RO
D000595:Amino Acid Sequence; D053318:Apolipoprotein E3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli; D006801:Humans; D047668:Inteins; D007553:Isotope Labeling / Q000379:methods; D008024:Ligands; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D053318:Apolipoprotein E3; D008024:Ligands; D009587:Nitrogen Isotopes; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins