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Enhanced Immunoaffinity Purification of Human Neutrophil Flavocytochrome B for Structure Determination by Electron Microscopy

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NADPH Oxidases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1982))

Abstract

Determination of the structure of human neutrophil (PMN) flavocytochrome b (Cytb) is a necessary step for the understanding of the structure-function essentials of NADPH oxidase activity. This understanding is crucial for structure-driven therapeutic approaches addressing control of inflammation and infection. Our work on purification and sample preparation of Cytb has facilitated progress toward the goal of structure determination. Here we describe exploiting immunoaffinity purification of Cytb for initial examination of its size and shape by a combination of classical and cryoelectron microscopic (EM) methods. For these evaluations, we used conventional negative-stain transmission electron microscopy (TEM) to examine both detergent-solubilized Cytb as single particles and Cytb in phosphatidylcholine reconstituted membrane vesicles as densely packed random, partially ordered, and subcrystalline arrays. In preliminary trials, we also examined single particles by cryoelectron microscopy (cryoEM) methods. We conclude that Cytb in detergent and reconstituted in membrane is a relatively compact, symmetrical protein of about 100 Å in maximum dimension. The negative stain, preliminary cryoEM, and crude molecular models suggest that the protein is probably a heterotetramer of two p22phox and gp91phox subunits in both detergent micelles and membrane vesicles. This exploratory study also suggests that high-resolution 2D electron microscopic approaches may be accessible to human material collected from single donors.

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Acknowledgments

We thank the Montana State University College of Letters and Sciences for funding the Sabbatical leave during the 2007–2008 academic year for AJJ during which the initial phases of this work were carried out. We also acknowledge the PHS grant 5R01AI26711 and the ARRA supplement for support during the subsequent period. Thanks also go to J. Quispe, B. Carragher, and C. Potter of the National Resource for Automated Molecular Microscopy at the Scripps Research Institute and S. Ludtke of the Baylor College of Medicine for help in obtaining and processing the cryoEM images. Lastly, special thanks go to the Thermal Biology Institute, NSF EPSCOR, and The Montana Nanotechnology Facility (MONT) for funding the negative-stain electron microscopy time.

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Author notes

  1. Ross M. Taylor

    Present address: Universal Cells , Seattle, WA, USA

Authors and Affiliations

  1. Department of Microbiology and Immunology, Montana State University, Bozeman, MT, USA

    Algirdas J. Jesaitis, Marcia Riesselman & Ross M. Taylor

  2. Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, USA

    Susan Brumfield

Authors
  1. Algirdas J. Jesaitis
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  2. Marcia Riesselman
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  3. Ross M. Taylor
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  4. Susan Brumfield
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Corresponding author

Correspondence to Algirdas J. Jesaitis .

Editor information

Editors and Affiliations

  1. Conway Institute, School of Medicine, University College Dublin, Dublin, Ireland

    Ulla G. Knaus

  2. Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Thomas L. Leto

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Jesaitis, A.J., Riesselman, M., Taylor, R.M., Brumfield, S. (2019). Enhanced Immunoaffinity Purification of Human Neutrophil Flavocytochrome B for Structure Determination by Electron Microscopy. In: Knaus, U., Leto, T. (eds) NADPH Oxidases. Methods in Molecular Biology, vol 1982. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9424-3_3

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  • DOI: https://doi.org/10.1007/978-1-4939-9424-3_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9423-6

  • Online ISBN: 978-1-4939-9424-3

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