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Characterizing metal-binding sites in proteins with X-ray crystallography

Nature Protocols volume 13, pages 10621090 (2018) | Download Citation

Abstract

Metals have crucial roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often a source of irreproducibility caused by issues such as pH changes, incorporation of unexpected metals, or oxidization/reduction of the metal. This protocol outlines the guidelines and best practices for characterizing metal-binding sites in protein structures and alerts experimenters to potential pitfalls during the preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence (XRF) spectra, and collecting diffraction data sets above and below the corresponding metal absorption edges. This protocol should allow experimenters to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecule samples as described in this protocol should enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within a period of 7–190 d, most of which (2–180 d) is devoted to growing the crystal. The protocol should be readily understandable to structural biologists, particularly protein crystallographers with an intermediate level of experience.

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Acknowledgements

This work was supported by federal funds awarded to W.M. from the National Institute of General Medical Sciences under grant numbers GM117325 and GM117080, and NIH BD2K grant HG008424, as well as from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Department of Health and Human Services under contract nos. HHSN272201200026C and HHSN272201700060C. E.N. was supported by the Foundation for Polish Science (FNP) and received funding from the Marian Smoluchowski Krakow Research Consortium—a Leading National Research Centre KNOW supported by the Polish Ministry of Science and Higher Education. We thank J. Lipowska for providing the fluorescence and diffraction data for dihydroorotase from Y. pestis CO92. We thank M.P. Czub for providing Thermofluor shift data for STM1931 protein from S. Typhimurium. We thank R. Alkire (Structural Biology Center at Argonne National Laboratory) for providing the fluorescence spectrum data for the zinc foil. We thank D.R. Cooper and M. Cymborowski for help with XRF data collection and interpretation. We thank M. Grabowski, W.-S. Tzou, and B. Venkataramany for valuable discussions and critical reading of the manuscript.

Author information

Author notes

    • Katarzyna B Handing
    • , Ewa Niedzialkowska
    •  & Ivan G Shabalin

    These authors contributed equally to this work.

Affiliations

  1. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA.

    • Katarzyna B Handing
    • , Ewa Niedzialkowska
    • , Ivan G Shabalin
    • , Heping Zheng
    •  & Wladek Minor
  2. Center for Structural Genomics of Infectious Diseases (CSGID), University of Virginia, Charlottesville, Virginia, USA.

    • Katarzyna B Handing
    • , Ewa Niedzialkowska
    • , Ivan G Shabalin
    • , Heping Zheng
    •  & Wladek Minor
  3. Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Krakow, Poland.

    • Ewa Niedzialkowska
  4. Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California, USA.

    • Misty L Kuhn

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Contributions

K.B.H. performed essential experiments to identify the pH-dependent conformational changes of residues coordinating the transition metal in albumin; E.N. provided important data and experience in ICP–OES and TSA experiments; K.B.H., I.G.S., E.N., M.L.K., and H.Z. provided critical experience in metal-binding-protein production and purification; K.B.H., I.G.S., E.N., and W.M. provided critical experience in metal-binding-protein crystallization and data collection; H.Z. and I.G.S. provided extensive experience in characterization of metal-binding sites in protein structures; H.Z. and I.G.S. laid out the structural framework of the manuscript; M.L.K. provided constructive comments and extensively edited the manuscript; K.B.H., E.N., I.G.S., M.L.K., H.Z., and W.M. wrote and approved the manuscript; and H.Z. and W.M. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Heping Zheng or Wladek Minor.

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1 and 2, and Supplementary Tables 1–4

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DOI

https://doi.org/10.1038/nprot.2018.018

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