Role of charged amino acid side chains in the stability and lipid binding ofManduca sexta apolipophorin III

Chemical modification procedures were employed to neutralize charged amino acid side chains of apolipophorin III (apoLp-III). Glutamate plus aspartate carboxylate side chains were amidated while, in other experiments, the epsilon-amino groups of lysine residue side chains were acetylated. Circular dichroism (CD) spectroscopy was performed to assess the effect of chemical modification on the secondary structure of apoLp-III. Compared to control, unmodified apoLp-III, both amidated and acetylated apoLp-IIIs possessed significantly diminished levels of alpha-helical structure. A similarly significant amount of alpha-helix structure could be induced in both modified apoLp-IIIs, however, by the addition of 50% trifluoroethanol, a helix inducing solvent, indicating that the proteins have retained their capacity to form helical secondary structures. The lipid binding interactions of chemically modified apoLp-IIIs were also examined in lipoprotein binding assays. Whereas control, unmodified apoLp-III displayed lipid binding activity, neither modified apoLp-III was capable of interaction with the substrate lipid surface. In phospholipid binding assays using the model compound, dimyristoylphosphatidylcholine, acetylated apoLp-III failed to interact while amidated apoLp-III showed limited interaction. When sodium dodecyl sulfate (SDS) micelles were employed as a model lipid surface, interaction of the modified apoLp-IIIs was observed. To characterize the relative stability of the interaction of control and modified apoLp-IIIs with SDS micelles, urea denaturation studies were performed. These experiments showed that, while control and amidated apoLp-IIIs were relatively resistant to urea induced denaturation, acetylated apoLp-III was susceptible. Taken as a whole, the results suggest that charged amino acid residues play an important role in stabilization of the lipid-free helix bundle conformation of apoLp-III and may promote stabilization of the lipid bound state through charge-charge interactions with lipoprotein surface components. (C) 1995 Wiiey-Liss, Inc.