Researcher(s)
- Sydney Ziemba, Biochemistry, University of Delaware
Faculty Mentor(s)
- Jeffrey Mugridge, Department of Chemistry and Biochemistry, University of Delaware
Abstract
AlkBH4, a member of the AlkB family of homologues, has been found to be overexpressed in non-small-cell lung cancer in humans and its expression level has been correlated with the long-term prognosis in these patients. It has been suggested that AlkBH4 helps regulate actin–myosin interaction by catalyzing the demethylation of monomethylated K84 on actin, and that AlkBH4 plays a role in transcriptional silencing by removing the methyl group modification on N6-methyladenosine on double stranded DNA. Additionally, it has been proposed that AlkBH4 affects protein translation by installing an (R)-5-carboxyhydroxymethyluridine ((R)-mchm5U) modification at the wobble position of 5-methoxycarbonylmethyluridine (mcm5U) modified tRNAs.
Structural characterization would provide insight into how AlkBH4 is able to accommodate its several very distinct substrates in its active site. However, the structure of AlkBH4 has not previously been solved. Prior attempts at crystallizing AlkBH4 on its own, for use in X-ray crystallography, have been unsuccessful. Two different truncations were designed to remove a predicted N-terminal disordered region that may be hindering crystal formation. Additionally, lysozyme-AlkBH4 and maltose-binding protein (MBP)-AlkBH4 fusion protein constructs were designed to enhance crystallizability by providing additional surface area.
The fusion proteins have been overexpressed in E. coli BL21 cells, and will be purified for use in crystallization. The Δ21 truncation of AlkBH4 has been overexpressed in E. coli BL21 cells, and purified by NiNTA affinity chromatography, anion exchange chromatography, and size exclusion chromatography. The purified protein has been screened against different crystallization conditions from commercial high-throughput screens, and crystal hits have been identified. The conditions that produced these hits will be optimized to produce better quality crystals. In the future, these crystals will be sent to national laboratory beamlines for X-ray diffraction, followed by structural analysis of high-resolution datasets.