Apolipoprotein A-I (apoA-I) is the central protein of high-density lipoprotein (HDL) and is comprised of two domains; a highly ordered N-terminal domain and a less structured C-terminal (CT) domain. The CT domain mediates self-association in the lipid free state, and initiates lipid binding leading to HDL formation. The CT domain has six lysine residues at position 195, 206, and 208 in helix 8 and 226, 238, and 239 in helix 10. Substitution of all six lysine residues with glutamine resulted in a monomeric form of apoA-I. To identify lysines critical for self-association, nine single and two triple mutants were engineered via site-directed mutagenesis. Circular dichroism and chemical denaturation revealed the mutants retained their structural integrity. Chemical crosslinking and size exclusion chromatography demonstrated the helix 10 triple mutant was predominantly monomeric, while the helix 8 triple mutant remained an oligomer. All helix 10 single mutants displayed moderately reduced self-association, suggesting these lysines work collectively to promote self-association. The helix 10 triple mutant transformed phospholipid vesicles at a higher rate than the wild-type protein suggesting the monomeric protein may solubilize phospholipids more efficiently. When helix 10 glutamates 223, 234, and 235 were substituted with glutamine they displayed similar degrees of self-association to that of the lysine single mutants, suggesting similar ionic interactions. The results of this study indicate that intrahelical salt-bridges play a prominent role in apoA-I oligomerization through stabilization of amphipathic α-helices. Assembly of the helix 10 helices from neighboring proteins likely results in an intermolecular helix bundle thereby promoting protein oligomerization.
|Commitee:||Narayanaswami, Vasanthy, Schwans, Jason|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 82/6(E), Masters Abstracts International|
|Keywords:||C-terminal ionic residues, Apolipoprotein A-I|
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