Role of Buried Polar Residues in Helix Bundle Stability and Lipid Binding of Apolipophorin III: Destabilization by Threonine 31

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Abstract
Apolipophorin III (apoLp-III) from Locusta migratoria is a model exchangeable apolipoprotein that plays a key role in neutral lipid transport. The protein is comprised of a bundle of five amphipathic α-helices, with most hydrophobic residues buried in the protein interior. The low stability of apoLp-III is thought to be crucial for lipid-induced helix bundle opening, to allow protein−lipid interactions. The presence of polar residues in the hydrophobic protein interior may facilitate this role. To test this, two buried polar residues, Thr-31 and Thr-144, were changed into alanine by site-directed mutagenesis. Secondary structure analysis and GdnHCl- and temperature-induced denaturation studies indicated an increase in α-helical content and protein stability for T31A apoLp-III compared to wild-type apoLp-III. In contrast, T144A had a decreased α-helical content and protein stability, while tryptophan fluorescence indicated increased exposure of the hydrophobic interior to buffer. Two mutant proteins that had lysine residues introduced in the hydrophobic core displayed a more pronounced decrease in secondary structure and protein stability. Lipid binding studies using phospholipid vesicles showed that T31A apoLp-III was able to transform phospholipid vesicles into discoidal particles but at a 3-fold reduced rate compared to wild-type apoLp-III. In contrast, the less stable apoLp-III mutants displayed an increased ability to transform phospholipid vesicles. These results demonstrate the inverse correlation between protein stability and the ability to transform phospholipid vesicles into discoidal protein−lipid complexes and that Thr-31 is a key determinant of the relatively low protein stability, thereby promoting apoLp-III to interact with lipid surfaces.