Abstract 417: Structural Characterization of 20 kDa Fragments of Apolipoprotein B100
Apolipoprotein (apo) B100 is a 550 kDa hydrophobic glycoprotein that forms the structural backbone for the assembly of triglyceride-rich lipoproteins. ApoB100 circulates bound to the same lipoprotein particle on which it is synthesized (i.e. it is non-exchangeable) until the lipoprotein is cleared from the plasma via receptor-mediated uptake. The polypeptide length and aqueous insolubility are considerable obstacles to the determination of apoB100 structure. Based on primary structure algortihms, apoB100 is predicted to contain 5 domains, comprising an N-terminal globular mixed βα1 domain followed by alternating amphipathic β-strand and α-helix rich regions (N-βα1-β1-α2-β2-α3-C). Previous studies from our laboratory have shown that amino acids 1694-1880 (B1) of the β1 domain of apoB100 bind tightly to triglyceride droplets in vitro. We have expressed this 20 kDa protein fragment, and a second 20 kDa fragment B2 (amino acids 1881-2070) that lies at the junction between the β1 and α2 domains, in bacterial cell culture as hexahistidine-tagged fusion proteins. B1 was insoluble in aqueous solution, but soluble in the presence of 150 mM dodecylphosphocholine (DPC), whereas B2 was soluble in aqueous solution, with or without DPC. Circular dichroism (CD) spectroscopy of DPC-solubilized B1 and B2 fragments indicated an approximately equal proportion of α-helix, β-strand/β-turn and unordered structures in B1 (36%, 37% and 28%, respectively) vs. approximately 56% α-helix, 27% β-strand/β-turn and 17% unordered content for B2. Preliminary NMR spectroscopy of 13C/15N labeled B1 has been conducted. Estimates of protein secondary structure from backbone chemical shift assignments using the algorithm DANGLE (Dihedral ANgles from Global Likelihood Estimates) suggested significant α-helical character in B1. These studies suggest that the α-helical content may be underestimated by CD, and generation of a high-resolution structure of B1 is in progress. Our studies have provided the first atomic-level structural information for structural elements of apoB100 and suggest a central role for unique amphipathic structures in lipoprotein structure and function.
Author Disclosures: A.M. Reda: None. M.L. Tremblay: None. J.K. Rainey: None. R.S. McLeod: None.
- © 2014 by American Heart Association, Inc.