Tryptophan Probes at the Apolipoprotein C-III and Membrane Interface

Abstract

Lipoproteins are lipid-protein complexes which facilitate lipid transport throughout the body. The proteins which bind lipids to form these complexes are called apolipoproteins. Since apolipoproteins play essential roles in lipid metabolism, their dysfunction is associated with numerous diseases. Therefore, it is vital to gain a detailed molecular understanding of their interactions with lipid. In this work, we investigate the site-specific membrane interactions of apolipoprotein C-III (apoC-III) by measuring the steady-state fluorescence and time-resolved anisotropy of single Trp containing variants (W54F/W65F (W42), W42F/W65F (W54), W42F/W54F (W65)) in the presence of sodium dodecyl sulfate (SDS) micelles and phospholipid vesicles (1:1 molar ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)). Upon addition of vesicles or SDS micelles, apoC-III undergoes a secondary structural change from disordered to α-helical as indicated by circular dichroism (CD) spectroscopy. Using CD and fluorescence data, apparent membrane partition constants were extracted. Though secondary structural formation is comparable amongst the Trp variants upon vesicle binding, distinct site-specific Trp environments are observed. The penetration depths of W42, W54, and W65 into the vesicle bilayer also were assessed by using lipids labeled with the heavy-atom quencher, bromine, at different positions in the lipid hydrocarbon acyl-chain.