Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion: Q-XAD

Abstract

Edited by M. Schiltz, Fonds National de la Recherche, Luxembourg Keywords: metal quantification; quantitative X-ray anomalous dispersion; R2-like ligandbinding oxidase; Q-XAD; X-ray crystallography.Supporting information: this article has supporting information at journals.iucr.org/dHere, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a heterodinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of highquality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided. research papers Acta Cryst. (2019). D75, 764-771 Griese & Hö gbom Location-specific quantification of metal ions 765

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins

“…Both scaled and unscaled datasets were analyzed. Anomalous difference maps were calculated with PHENIX (57) using the phases from a ligand-free model, and the relative amounts of Fe and Mn in each metal site were calculated as previously described (60). The intensities of the anomalous difference density peaks in spheres of 1.9 Å radius around the center of the peaks were integrated using MAPMAN (61).…”

The Bacillus anthracis class Ib ribonucleotide reductase subunit NrdF intrinsically selects manganese over iron

“…In addition, we have introduced an R2lox-like L61G mutation in the second coordination sphere in close proximity to the N-terminal metal binding site (site 1) and investigated its effect on the structure and protein metallation. The locationspecific metal content of metal-loaded proteins was analyzed by Q-XAD [37]. To ensure that the protein was metal-free prior to cofactor reconstitution, WT and the variant were analyzed by TXRF.…”

“…Analysis of anomalous diffraction data and metal quantification were done essentially as described in ref. [37]. Data were integrated with DIALS [42].…”

“…Here, we use quantitative X-ray anomalous dispersion (Q-XAD) [37] to show that the BaR2b protein scaffold has an intrinsic metal specificity allowing it to select Mn over Fe in both metal binding positions. This observation supports the assignment of di-manganese as the biologically relevant cofactor in vivo.…”

“…At the Fe edge, both Mn and Fe display an anomalous signal. Quantification of metal content in each position is done via peak integration of the respective anomalous difference maps (Q-XAD[37])…”