MitoNEET, a [2Fe-2S] cluster protein, has the capability to bind to the antidiabetic drug pioglitazone. Possible cellular functions of mitoNEET include as an electron-transport protein or an iron-sulfur transfer protein. The ligation of the [2Fe-2S] cluster of mitoNEET is unique in using one histidine and three cysteine residues. Recombinant expression of mitoNEET was accomplished using e. Coli bacteria and was subsequently purified by using affinity fast protein liquid chromatography. After expression and purification, UV-Visible spectroscopy is used to observe the local environment of the cluster through observation of the ligand to metal charge transfer (LMCT) bands. MitoNEET was reacted with the chemical modifier diethylpyrocarbonate (DEPC) using a range of pH values. DEPC reacts with histidine to form a chromophore at 250 nm. However, a change in the LMCT bands after DEPC addition indicates modification of His87, the ligating histidine of mitoNEET. The goal of chemical modification of mitoNEET is to investigate how the state of His87 impacts protein stability and chemical reactivity towards small molecules like pioglitazone and macromolecules like protein binding partners of mitoNEET.

Expression, purification, and chemical modification of MitoNEET: A novel diabetes drug target

246th National Meeting (2013)

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