STRUCTURAL CHARACTERIZATION OF 20 KDA LIPID-BINDING FRAGMENTS OF APOLIPOPROTEIN B100

Abstract

Apolipoprotein(apo)B100 is a 550 kDa hydrophobic glycoprotein that forms the structural backbone for the assembly of triglyceride(TG)-rich lipoproteins. ApoB100 is predicted to contain 5 domains: an N-terminal globular βα1 domain followed by alternating amphipathic β-strand and α-helical regions (N-βα1-β1-α2-β2-α3-C). However, the hydrophobicity and size of apoB100 have prevented experimental verification of this structural prediction. Amino acids 1694-1880 (B1), within the β1 domain, tightly bind TG in vitro. We expressed and purified two 20 kDa fragments, B1 and B2 (amino acids 1881-2070, which lies at the junction between the β1 and α2 domains), in bacterial culture in order to collect high resolution structural information. Under conditions ensuring no more than one apoB per micelle, circular dichroism (CD) spectroscopy of B1 and B2 in dodecylphosphocholine (DPC) or micelles formed with three different lysophospholipids indicated predominantly α-helical character. NMR spectroscopy of 13C/15N-labeled B1 and B2 in DPC micelles also indicated substantial α-helical structure. Our studies provide the first atomic-level evidence for amphipathic α-helical structural elements in the TG-binding regions of apoB100.