Metals in enzyme catalysis and visualization methods
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Metal ions play essential roles in biological functions including catalysis, protein stability, DNA-protein interactions and cell signaling. It is estimated that 30% of proteins utilize metals in some fashion. Additionally, methods by which metal ions can be visualized have been utilized to study metal concentrations and localizations in relation to disease. Understanding the roles metals play in biological systems has great potential in medicine and technology. Chapters 1 and 2 of this dissertation analyzes the structure and function of the Mn-dependent enzyme oxalate decarboxylase (OxDc) and Chapter 2 presents a bioinformatic analysis of the cupin superfamily that provides the structural scaffold of the decarboxylase. The X-ray crystal structure of the W132F variant was determined and utilized together with EPR data to develop a computational approach to determining EPR spectra of the enzyme’s two metal-binding centers. Furthermore, a variant in which the catalytic Glu162 was deleted revealed the binding mode of oxalate, the first substrate-bound structure of OxDc. OxDc is a member of the cupin superfamily, which comprises a wide variety of proteins and enzymes with great sequence and functional diversity. A bioinformatics analysis of the superfamily was performed to analyze how sequence variation determines function and metal utilization. Chapters 3 and 4 discuss the expansion of lanthanide-binding tags (LBTs) to in cellulo studies. Lanthanide-binding tags are short sequences of amino acids that have high affinity and selectivity for lanthanide ions. An EGF-LBT construct used to quantify EGF receptors on the surface of A431 and HeLa cells. The results from the LBT quantification are consistent with previous studies of EGFR receptors in these cell types, validating the use of this method for future studies. The potential of using LBTs for X-ray fluorescence microscopy (XFM) was also investigated. LBT-labeled constructs were utilized to investigate if membrane bound as well as cytosolic LBT-containing proteins could be visualized and localized to their cell compartments via XFM; both membrane-localized and cytosolic proteins were successfully visualized. With the high resolution (< 150 Å) obtainable with new synchrotron beamline configurations LBTs could be used to study nanoscale biological structures in their near-native state.