The N-Terminus of Apolipoprotein A-V Adopts a Helix-Bundle Molecular Architecture

Previous studies of recombinant full-length human apolipoprotein A-V (apoA-V) provided evidence of the presence of two independently folded structural domains. Computer-assisted sequence analysis and limited proteolysis studies identified an N-terminal fragment as a candidate for one of the domains. C-Terminal truncation variants in this size range, apoA-V(1-146) and apoA-V(1-169), were expressed in Escherichia coli and isolated. Unlike full-length apoA-V or apoA-V(1-169), apoA-V(1-146) was soluble in neutral-pH buffer in the absence of lipid. Sedimentation equilibrium analysis yielded a weight-average molecular weight of 18811, indicating apoA-V(1-146) exists as a monomer in solution. Guanidine HCl denaturation experiments at pH 3.0 yielded a one-step native to unfolded transition that corresponds directly with the more stable component of the two-stage denaturation profile exhibited by full-length apoA-V. On the other hand, denaturation experiments conducted at pH 7.0 revealed a less stable structure. In a manner similar to that of known helix bundle apolipoproteins, apoA-V(1-146) induced a relatively small enhancement in 8-anilino-1-naphthalenesulfonic acid fluorescence intensity. Quenching studies with single-Trp apoA-V(1-146) variants revealed that a unique site predicted to reside on the nonpolar face of an amphipathic R-helix was protected from quenching by KI. Taken together, the data suggest the 146 N-terminal residues of human apoA-V adopt a helix bundle molecular architecture in the absence of lipid and, thus, likely exist as an independently folded structural domain within the context of the intact protein

Apolipoprotein A–I binding to anionic vesicles and lipopolysaccharides: Role for lysine residues in antimicrobial properties

AbstractHuman apolipoprotein A–I (apoA–I) is a 28kDa protein and a major component of high-density lipoproteins, mediating several essential metabolic functions related to heart disease. In the present study the potential protective role against bacterial pathogens was explored. ApoA–I suppressed bacterial growth of Escherichia coli and Klebsiella pneumoniae. The protein was able to bind lipopolysaccharides and showed a strong preference for bilayer vesicles made of phosphatidylglycerol over phosphatidylcholine. Lysine side chains of apoA–I were acetylated to evaluate the importance of electrostatic forces in the binding interaction with both membrane components. Electrophoresis properties, dot blot analysis, circular dichroism, and fluorescence spectroscopy to probe for changes in protein structure indicated that the acetylated protein displayed a strongly reduced lipopolysaccharide and phosphatidylglycerol binding. A mutant containing only the N-terminal domain of apoA–I also showed a reduced ability to interact with the membrane components, although to a lesser extent. These results indicate the potential for apoA–I to function as an antimicrobial protein and exerts this function through lysine residues