NMR evidence for a conformational adaptation of apolipophorin III upon lipid association

A characteristic property of amphipathic exchangeable apolipoproteins is an ability to exist alternately in lipid-free and lipid-bound states. In the present study, we have used H-1-N-15-heteronuclear single quantum correlati on spectroscopy to probe structural changes of apolipophorin III upon lipid association, by monitoring alterations of the chemical shifts of specific amino acids as a function of lipid titration. N-15-valine-, N-15-leucine-, N-15-lysine-, and N-15-glycine-labeled apolipophorin III were used in titra tion experiments with the micelle-forming lipid dodecylphosphocholine. In t he absence of lipid, valine and leucine residues are located in the hydroph obic interior of the apolipophorin TIT helix bundle and their resonances re sist chemical shift changes below the critical micelle concentration of dod ecylphosphocholine. At the critical micelle concentration, however, dramati c and abrupt chemical shift changes occur, apparently coincident with forma tion of a protein-lipid micelle complex, as judged by significant line-widt h broadening of the crosspeaks. By contrast, apolipophorin III lysine and g lycine residues are located on the hydrophilic surfaces of amphipathic alph a-helices or in loop regions, exposed to solvent. Their crosspeaks display either a chemical shift change similar to that seen for hydrophobic residue s or a more gradual chemical shift change, beginning at very low dodecylpho sphocholine concentrations. These results indicate that an interaction occu rs between specific solvent-exposed lysine residues and dodecylphosphocholi ne below the critical micelle concentration of this lipid, whereas valine a nd leucine residues are not accessible to monomeric dodecylphosphocholine. At the critical micelle concentration, however, the availability of a newly formed lipid surface induces apolipophorin III binding, concomitant with c onformational opening of the helix bundle, exposing its hydrophobic surface s for binding to the dodecylphosphocholine micellar surface. Subsequently, hydrophobic residues undergo characteristic spectral changes. Subtle differ ences in behavior of specific hydrophobic residues, in terms of their respo nse to dodecylphosphocholine titration and relative locations in the helix- bundle conformation, suggest that one end of the molecule may initiate cont act with the lipid surface, followed by helix bundle opening.