Pyrene fluorescence analysis offers new insights into the conformation of the lipoprotein-binding domain of human apolipoprotein E.

The C-terminal domain (CT) of apolipoprotein E (apoE), a critical protein involved in cholesterol transport in the plasma and brain, plays an important role in high-affinity lipoprotein binding. Although high-resolution structural information is available for the N-terminal domain of apoE, the structural organization of the CT (residues 201-299) is largely unknown. In this study, we employ site-specific fluorescence labeling with pyrene maleimide to gain insight into the structure and conformation of apoE CT in its naturally self-associated state in buffer at physiologically relevant concentrations (5-50 microg/mL). Pyrene is a highly sensitive fluorophore that reports on spatial proximity between desired sites by displaying unique spectral features. Pyrene was covalently attached to single cysteine-containing recombinant human apoE CT at position 223 or 255 to probe the first predicted helical segment and at position 277 to monitor the terminal predicted helical segment. Regardless of the location of the probe, all three pyrene-labeled apoE CT variants display an intense and dramatic fluorescence excimer band at 460 nm, a signature feature of pyrene, which indicates that two pyrene moieties are within 10 A of each other. In addition, an intense peak at 387 nm (indicative of a highly hydrophobic environment) was noted in all cases. Fluorescence emission quenching by potassium iodide indicates that the accessibility to the probes was restricted at these locations. The possibility that the hydrophobicity of the pyrene moiety was the driving force for helix-helix interaction was excluded because pyrene located at position 209, which is predicted to be located in a nonhelical segment, did not display the above intense unique features. Lastly, denaturation studies suggest that the terminal helix unfolds prior to the first predicted helix in apoE CT. Our studies indicate that there are extensive intermolecular helix-helix contacts throughout the entire CT in the lipid-free state with two apoE CT molecules oriented parallel to each other to form a dimer, which dimerizes further to yield a tetramer. Such an organization allows helix-helix interactions to be replaced by helix-lipid interactions upon encountering a lipoprotein surface, with the terminal helix likely initiating the binding interaction. This study presents the possibility of employing pyrene fluorophores as powerful new alternatives to obtain conformational information of proteins at physiologically relevant concentrations.