Dynamics and hydration of the alpha-helices of apolipophorin III.
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.
Soulages
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JL
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA. jose@biochem.okstate.edu
Arrese
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EL
D019943:Amino Acid Substitution; D000818:Animals; D001053:Apolipoproteins / Q000737:chemistry*; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D006110:Grasshoppers; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan
D001053:Apolipoproteins; D011994:Recombinant Proteins; C042470:apolipophorin III; D014364:Tryptophan