Enhanced Immunoaffinity Purification of Human Neutrophil Flavocytochrome B for Structure Determination by Electron Microscopy
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.
Key wordsimmunoaffinity purification NOX2 flavocytochrome b electron microscopy cryoEM 2D membrane reconstitution negative stain mAb CS9
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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