Apolipophorin Ill (apoLp-lll) from the insect Locusta migratoria is a model apolipoprotein to study their structure-function relationship. In contrast to the common four-helix bundle motif, apoLp-III is a bundle of five amphipathic α-helices (H1-H5). It was hypothesized that H1-H5 of apoLp-III may have evolved to modulate lipid binding. To verify this, N-terminal (H2-H5) and C-terminal (H1-H4) helix deletion mutants were designed. H2-H5 was expressed in a bacterial expression system. Since H1-H4 could not be expressed, a methionine mutant was designed allowing removal of helix 5 by CNBr digestion. Five additional truncation mutants apoLp-III (1-135, 1-139, 1-143, 1-149 and 1-153) were designed by stop codon insertion to determine the region in helix 5 required for protein expression. Circular dichroism and guanidine-HCl denaturation analysis showed a significantly lower α-helical content and stability for all mutants compared to wild type apoLp-III. However, a substantially higher lipid binding was observed for all mutant proteins.
|Advisor:||Weers, Paul M. M.|
|Commitee:||McAbee, Douglas D., Narayanaswami, Vasanthy, Weers, Paul M. M.|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 51/04M(E), Masters Abstracts International|
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