Tryptophan residues stabilize the N-terminal domain of apolipoprotein A-I and promote its anchoring to lipid surfaces

Background. All the Trp residues of plasma apoA-I and recombinant proapoA-I are located in the N-terminal half of the proteins. Thus, these aromatic residues are useful probes of the N-terminal domain organization of apoA-I, its stability and its interaction with lipids. Methods. Four Trp --> Phe mutants of proapoA-I, with substitutions of two and four Trp residues, were prepared by PCR mutagenesis and expression in Escherichia coli. The purified proteins were studied by circular dichroism, fluorescence and absorbance methods in lipid-free and lipid-bound forms. Results and Conclusions. The Trp --> Phe mutants had very similar structures to wild-type proapoA-I, both in the lipid-free state and in reconstituted HDL. However, the lipid-free mutants with increasing contents of Phe residues had progressively lower free energies of unfolding, indicating that Trp side chains are important in stabilizing the N-terminal domain of apoA-I in solution. In addition, the Trp --> Phe mutants cleared phospholipid liposomes at lower rates than proapoA-I, suggesting that Trp residues in the N-terminal domain participate in the initial anchoring of apoA-I to lipid surfaces.