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Initiation of lipid binding in Locusta migratoria apolipophorin III
Author(s) -
Phung Yin Mei,
Rodriguez Johana,
Marti Daisy,
Abdullahi Gezman,
Weers Paul M. M.
Publication year - 2010
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.24.1_supplement.478.3
Subject(s) - helix (gastropod) , helix bundle , circular dichroism , mutant , protein secondary structure , alpha helix , biophysics , biochemistry , triple helix , protein folding , chemistry , isothermal titration calorimetry , denaturation (fissile materials) , helix turn helix , protein structure , collagen helix , biology , dna binding protein , stereochemistry , transcription factor , ecology , nuclear chemistry , snail , gene
Apolipophorin III (apoLp‐III) from Locusta migratoria is an exchangeable apolipoprotein used as a model to study the structure and function of apolipoproteins. The NMR solution structure of apoLp‐III shows a 5‐helix bundle with an additional 4‐residue helix (helix 4’) located between helix‐4 and helix‐5. We hypothesize that this mini helix initiates lipid binding. An alternative mechanism is that the 1 st or 5 th helix makes the initial contact with the lipid surface. To test both mechanisms, a site‐directed mutagenesis approach was employed. Helix 4’ was converted into a type I β‐turn, and in separate single mutants Trp‐130 was changed into Gln and Ala‐131 into Ser. In addition, two 4‐helix apoLp‐III mutants were designed by deleting the coding regions for helix‐1 or helix‐2. Based on DNA sequencing, the first round of mutagenesis produced the two single mutations in helix 4’ and the mutant without helix‐1. These three mutant proteins were successfully expressed and purified from E. coli . However, no recombinant protein was found in E. coli for the type I β‐turn and the helix‐5 deletion mutants. Circular dichroism and guanidine‐HCl denaturation analysis will be used to assess the secondary structure and stability of the mutant proteins. Changes in the lipid binding properties will be studied using phospholipid liposomes and binding parameters will be determined by isothermal calorimetry.