Single tryptophan mutants of apolipophorin III: binding interaction to lipopolysaccharides

Abstract

Apolipoproteins have potential to neutralize lipopolysaccharide (LPS) toxicity. To gain insight in this binding interaction, apolipophorin III (apoLp‐III) from the Greater wax moth, Galleria mellonella, was used as a model. Single tryptophan mutants of apoLp‐III were engineered to determine binding interaction with the three components of LPS (Lipid A, core and O‐antigen polysaccharides). Five single‐trytophan mutants (F20W, L80W, L119W, I138W and F145W) were designed and produced in a bacterial expression system. Based on circular dichroism analysis, the secondary structure of the mutant proteins was similar to that of the wild‐type protein. The protein stability, measured as the resistance to guanidine‐HCl induced denaturation, was slightly decreased. This indicates that the overall a‐helical fold was maintained in the mutant proteins. Tryptophan fluorescence spectroscopy was used to analyze the LPS binding interaction. Binding to LPS resulted in an increase in tryptophan fluorescence emission intensity for I138W and F145W‐apoLp‐III, a decrease for F20W and L119W‐apoLp‐III, and no significant change was observed for L80W‐apoLp‐III. Thus the tryptophan residues relocate into distinct environments, indicating that the apoLp‐III helices bind to different parts of LPS.