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Conformational change of apolipophorin III upon lipopolysaccharide binding
Author(s) -
Idangodage Thanuki Thivanka,
Idangodage Hasitha,
Weers Paul M.M.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a914-d
Subject(s) - mutant , cysteine , biochemistry , helix bundle , biophysics , conformational change , chemistry , helix (gastropod) , apolipoprotein b , biology , protein structure , microbiology and biotechnology , cholesterol , gene , ecology , snail , enzyme
Apolipophorin III (apoLp‐III) from the Greater wax moth, Galleria mellonella , is an exchangeable apolipoprotein. ApoLp‐III functions in the stabilization of low‐density lipophorin particles which are used to mobilize lipids. The protein is also able to bind to lipopolysaccharides (LPS), and may function as a pattern recognition protein. ApoLp‐III is organized as a five α‐helical bundle with a simple up‐and‐down topology. Lipid binding is accompanied by an opening of the helix bundle. We hypothesize that a similar conformational change in apoLp‐III occurs upon LPS binding. To prevent helix bundle opening, four cysteine residues were introduced to allow formation of two disulfide bridges to tether the helices. Cysteine pairs were engineered at position 5 on Helix (H) 1 and at position 135 on H5, and at position 37 on H2 and position 95 on H3 (P5C/A135C/N37C/E95C apoLp‐III). Mutant proteins were expressed in a bacterial _expression system, and purified by HPLC. Binding studies, using intrinsic tyrosine fluorescence and gel shift assays will be used to address the helix opening hypothesis. Proteins will be studied under oxidizing and reducing conditions to control helix bundle opening. Two other mutant apolipoproteins that have been shown to display decreased lipid binding will also be tested. These proteins, with mutations in the second loop, will be used to investigate the similarity between lipid and LPS binding.