Role of the N- and C-Terminal Helices in Apolipophorin III Stability and Lipid Binding

Abstract

Apolipophorin III (apoLp-III) from the insect Locusta migratoria is a model apolipoprotein to study their structure-function relationship. In contrast to the common 4-helix bundle motif, apoLp-III is a bundle of five amphipathic α-helices. We hypothesized that the 5-helix bundle of apoLp-III may have evolved to modulate lipid-binding activity. To verify this, N- and C-terminal helix deletion mutants were designed. The N-terminal deletion mutant was expressed and purified from E.coli. However, bacteria did not produce the C-terminal helix deletion mutant. Therefore, valine 131, which resides in the loop connecting helix-4 and −5, was replaced by methionine. Cleavage by CNBr then produced the C-terminal deletion variant. in addition, five truncation mutants in the C-terminal α-helix were engineered by introducing stop codons at Q136, E140, E144, Q150 and Q154, to create shortened versions of the protein. The mutant proteins truncated at E144, Q150 and Q154 were successfully expressed and purified but no protein was produced when apoLp-III was truncated at Q136 or E140. Guanidine denaturation analysis showed a decreased stability for the N- and C-terminal helix deletion mutants as indicated by the midpoint of denaturation (0.19 and 0.23 M for the deletion mutants and 0.50 M for wild-type apoLp-III). Truncation at E144, Q150 and Q154 also resulted in a decrease in protein stability with midpoints of 0.23, 0.35, and 0.36 M, respectively. Far UV circular dichroism showed a significant reduction in α-helical content for all mutants. Lipid binding analysis using dimyristoylphosphatidylcholine vesicles showed that the mutant proteins formed discoidal protein-lipid complexes at much higher rates compared to wild-type apoLp-III. The results show that deletion of a terminal α-helix result in an increased lipid binding but this comes at the cost of a decrease in protein stability.