Abstract 410: Binding of Apolipoprotein A-I to Inner and Outer Components of Bacterial Membranes

Abstract

Human apolipoprotein A-I (apoA-I) is a 28 kDa protein and a major component of high-density lipoproteins, mediating several essential metabolic functions related to heart disease. The protein is able to suppress bacterial growth of gram-negative bacteria. To understand the basis of the bacterial growth suppression and its potential as an antimicrobial protein, the binding interaction between apoA-I and membrane components of gram-negative bacteria was characterized using wild-type, modified, and a deletion variant of apoA-I. To modify the protein, lysine residues were acetylated with acetic anhydride. The deletion variant was created by removing the C-terminal amino acid residues 190-143. The binding interaction of apoA-I with lipopolysaccharides (LPS), abundantly present in the outer bacterial membrane of E. coli , was investigated with non-denaturing PAGE and isothermal titration calorimetry. In addition, protein structural changes induced by LPS binding were monitored by circular dichroism and fluorescence spectroscopy. Binding analysis showed that wild-type and N-terminal apoA-I were able to associate with LPS, but the acetylated protein showed a strongly reduced binding interaction. The bacterial inner membrane is rich in negatively charged phosphatidylglycerol. ApoA-I showed a strong preference for bilayer vesicles made of phosphatidylglycerol compared to phosphatidylcholine. Acetylated apoA-I showed a marked decrease in phosphatidylglycerol binding, while the binding activity of N-terminal apoA-I was only moderately decreased. These results indicated that apoA-I interacts with components of the inner and outer membrane of gram-negative bacteria. In addition, lysine side-chains play a crucial role in the binding interaction, indicating the importance of ionic interactions. These results indicate the potential for apoA-I to function as an antimicrobial protein.