title: Role of buried polar residues in helix bundle stability and lipid binding of apolipophorin III: destabilization by threonine 31.
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 alpha-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 alpha-helical content and protein stability for T31A apoLp-III compared to wild-type apoLp-III. In contrast, T144A had a decreased alpha-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.
authors.affiliation: Department of Chemistry and Biochemistry, California State University at Long Beach, Long Beach, California 90840, USA. pweers@csulb.edu
authors.forename: Paul M M
authors.initials: PM
authors.lastname: Weers
authors.forename: Wazir E
authors.initials: WE
authors.lastname: Abdullahi
authors.forename: Jamie M
authors.initials: JM
authors.lastname: Cabrera
authors.forename: Tzu-Chi
authors.initials: TC
authors.lastname: Hsu
mesh_terms: D019943:Amino Acid Substitution; D001053:Apolipoproteins / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017931:DNA Primers; D007700:Kinetics; D050356:Lipid Metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013912:Threonine
chemical_list: D001053:Apolipoproteins; D017931:DNA Primers; D011994:Recombinant Proteins; C042470:apolipophorin III; D013912:Threonine