Lanthanide-Binding Tags for 3D X-ray Imaging of Proteins in Cells at Nanoscale Resolution.

We report the application of lanthanide-binding tags (LBTs) for two- and three-dimensional X-ray imaging of individual proteins in cells with a sub-15 nm beam. The method combines encoded LBTs, which are tags of minimal size (ca. 15-20 amino acids) affording high-affinity lanthanide ion binding, and X-ray fluorescence microscopy (XFM). This approach enables visualization of LBT-tagged proteins while simultaneously measuring the elemental distribution in cells at a spatial resolution necessary for visualizing cell membranes and eukaryotic subcellular organelles.

X-ray Fluorescence Nanotomography of Single Bacteria with a Sub-15 nm Beam.

X-ray Fluorescence (XRF) microscopy is a growing approach for imaging the trace element concentration, distribution, and speciation in biological cells at the nanoscale. Moreover, three-dimensional nanotomography provides the added advantage of imaging subcellular structure and chemical identity in three dimensions without the need for staining or sectioning of cells. To date, technical challenges in X-ray optics, sample preparation, and detection sensitivity have limited the use of XRF nanotomography in this area. Here, XRF nanotomography was used to image the elemental distribution in individual E. coli bacterial cells using a sub-15 nm beam at the Hard X-ray Nanoprobe beamline (HXN, 3-ID) at NSLS-II. These measurements were simultaneously combined with ptychography to image structural components of the cells. The cells were embedded in small (3-20 µm) sodium chloride crystals, which provided a non-aqueous matrix to retain the three-dimensional structure of the E. coli while collecting data at room temperature. Results showed a generally uniform distribution of calcium in the cells, but an inhomogeneous zinc distribution, most notably with concentrated regions of zinc at the polar ends of the cells. This work demonstrates that simultaneous two-dimensional ptychography and XRF nanotomography can be performed with a sub-15 nm beam size on unfrozen biological cells to co-localize elemental distribution and nanostructure simultaneously.

Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase.

Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser(161)-Glu(162)-Asn(163)-Ser(164) in the N-terminal domain of OxDC with the cognate residues Asp(161)-Ala(162)-Ser-(163)-Asn(164) of an evolutionarily related, Mn-dependent oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity has now been investigated using membrane inlet mass spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C-C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild-type enzyme, even though the Glu(162) side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn(II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from an increased level of access of the solvent to the active site during catalysis, implying that the functional role of Glu(162) is to control loop conformation. A 2.6 Å resolution X-ray crystal structure of a complex between oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu(162) has been deleted from the active site loop, reveals the likely mode by which the substrate coordinates the catalytically active Mn ion prior to C-C bond cleavage. The "end-on" conformation of oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to mediate the formation of Mn(III) for catalysis upon substrate binding.

Assigning the EPR fine structure parameters of the Mn(II) centers in Bacillus subtilis oxalate decarboxylase by site-directed mutagenesis and DFT/MM calculations.

Oxalate decarboxylase (OxDC) catalyzes the Mn-dependent conversion of the oxalate monoanion into CO2 and formate. EPR-based strategies for investigating the catalytic mechanism of decarboxylation are complicated by the difficulty of assigning the signals associated with the two Mn(II) centers located in the N- and C-terminal cupin domains of the enzyme. We now report a mutational strategy that has established the assignment of EPR fine structure parameters to each of these Mn(II) centers at pH 8.5. These experimental findings are also used to assess the performance of a multistep strategy for calculating the zero-field splitting parameters of protein-bound Mn(II) ions. Despite the known sensitivity of calculated D and E values to the computational approach, we demonstrate that good estimates of these parameters can be obtained using cluster models taken from carefully optimized DFT/MM structures. Overall, our results provide new insights into the strengths and limitations of theoretical methods for understanding electronic properties of protein-bound Mn(II) ions, thereby setting the stage for future EPR studies on the electronic properties of the Mn(II) centers in OxDC and site-specific variants.