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Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins

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Oxygen Sensing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2648))

Abstract

Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein’s functional domain.

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Acknowledgments

This work was supported by grant 158120 from the Grant Agency of Charles University to J.V. Support from the EU and the Czech Ministry of Education, Youth, and Sport via the BioCeV (CZ.1.05/1.1.00/02.0109) and CIISB (LM2018127) projects is also gratefully acknowledged.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic

    Jakub Vávra, Artur Sergunin, Martin Stráňava, Toru Shimizu, Petr Man & Markéta Martínková

  2. Institute of Microbiology of the Czech Academy of Sciences, v.v.i., BIOCEV, Vestec, Czech Republic

    Alan Kádek & Petr Man

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  1. Jakub Vávra
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Correspondence to Petr Man or Markéta Martínková .

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  1. Department of Biochemistry and Molecular Biology, Penn State University, University Park, PA, USA

    Emily E. Weinert

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Vávra, J. et al. (2023). Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins. In: Weinert, E.E. (eds) Oxygen Sensing. Methods in Molecular Biology, vol 2648. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3080-8_8

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  • DOI: https://doi.org/10.1007/978-1-0716-3080-8_8

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