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The Importance of Salt‐Bridge Formation of Lysine 52 and 54 from Apolipophorin III for Protein Structure and Function
Author(s) -
Tran Angela,
Shah Kriti,
Weers Paul M.M.
Publication year - 2018
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.2018.32.1_supplement.792.37
Subject(s) - lysine , biochemistry , mutagenesis , mutant , biology , site directed mutagenesis , glutamine , chemistry , amino acid , gene
Apolipophorin III (apoLp‐III) is an 18 kDa exchangeable apolipoprotein found in insects. The protein is used as a model to better understand lipid transport processes and innate immunity due to the availability of three‐dimensional structures of the protein, allowing a structure‐guided approach. ApoLp‐III from Locusta migratoria contains eight lysine residues, which interact with negatively charged phospholipids found in bacterial membranes. They also help to maintain the structure of the protein by ionic interactions. Denaturation of the protein with guanidine‐HCl showed that when lysine residues at position 52 and 54 were changed into glutamine, protein stability was significantly reduced. To identify which of the two lysine residues are involved in salt bridge formation with neighboring glutamate or aspartate residues, site‐directed mutagenesis was employed to design single lysine to glutamine (KQ) variants. The K52Q and K54Q single mutants were generated using the Quick‐change site‐directed mutagenesis kit. E. coli cells were transformed with the mutant plasmids, and protein expression was induced by IPTG. SDS‐PAGE analysis of the bacterial cultures showed the presence of apoLp‐III in the media, indicating that the proteins escaped the bacteria. Upon purification by reversed‐phase HPLC the structure of the mutant proteins will be assessed using circular dichroism to determine helical content and protein stability, while phosphatidylglycerol bilayer vesicles will be used for functional studies. Identification of the salt‐bridge in apoLp‐III will help to better understand the role of ionic interactions in the structure and function of apolipoproteins. Support or Funding Information This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers GM089564, 8UL1GM118979‐02, 8TL4GM118980‐02, and 8RL5GM118978‐02. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .