Dynamics and Hydration of the α-Helices of Apolipophorin III

Jose L Soulages,Estela L Arrese

doi:10.1074/jbc.m909661199

Abstract

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein whose structure is represented as a bundle of five amphipathic alpha-helices. In order to study the properties of the helical domains of apolipophorin III, we designed and obtained five single-tryptophan mutants of Locusta migratoria apoLp-III. The proteins were studied by UV absorption spectroscopy, time-resolved and steady-state fluorescence spectroscopy, and circular dichroism. Fluorescence anisotropy, near-UV CD and solute fluorescence quenching studies indicate that the Trp residues in helices 1 (N-terminal) and 5 (C-terminal) have the highest conformational flexibility. These two residues also showed the highest degree of hydration. Trp residues in helices 3 and 4 display the lowest mobility, as assessed by fluorescence anisotropy and near UV CD. The Trp residue in helix 2 is protected from the solvent but shows high mobility. As inferred from the properties of the Trp residues, helices 1 and 5 appear to have the highest conformational flexibility. Helix 2 has an intermediate mobility, whereas helices 3 and 4 appear to constitute a highly ordered domain. From the configuration of the helices in the tertiary structure of the protein, we estimated the relative strength of the five interhelical interactions of apoLp-III. These interactions can be ordered according to their apparent stabilizing strengths as: helix 3-helix 4 > helix 2-helix 3 > helix 4-helix 1 approximately helix 2-helix 5 > helix 1-helix 5. A new model for the conformational change that is expected to occur upon binding of the apolipoprotein to lipid is proposed. This model is significantly different from the currently accepted model (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesemberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, M. (1991) Biochemistry 30, 603-608). The model presented here predicts that the relaxation of the tertiary structure and the concomitant exposure of the hydrophobic core take place through the disruption of the weak interhelical contacts between helices 1 and 5. To some extent, the weakness of the helix 1-helix 5 interaction would be due to the parallel arrangement of these helices.

Highlights

(3–5) and can be found associated with lipoproteins or in a lipid-free state
We have recently shown that the hydration and the tertiary interactions of Manduca sexta apoLp-III play a major role on the kinetics of association of this apolipoprotein with lipids [13]
The high mobility and hydration of the Trp residues located in these helices has prompted us to postulate a new potential mechanism for the conformational change that leads to the exposure of hydrophobic core of the apolipoprotein

Summary

Introduction

(3–5) and can be found associated with lipoproteins or in a lipid-free state. Several contributions to the understanding of the relationship between structure and function of exchangeable apolipoproteins have been made through the study of the insect exchangeable apolipoprotein, apolipophorin III (apoLpIII).1 Apolipophorin III is an exchangeable apolipoprotein (17 kDa) that is present as a free water-soluble protein, or bound to the lipid surface of the major insect lipoprotein, lipophorin (6 – 8). Fluorescence anisotropy, near-UV CD and solute fluorescence quenching studies indicate that the Trp residues in helices 1 (N-terminal) and 5 (C-terminal) have the highest conformational flexibility.

Results

Conclusion