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Hydrogen/Deuterium Exchange Behavior During Denaturing/Refolding Processes Determined in Tetragonal Hen Egg-White Lysozyme Crystals

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Abstract

The hydrogen/deuterium (H/D) exchange of main-chain amide hydrogens in the protein that denatured and refolded in deuterated solvent is considered to contain the traces of hydrogen bond cleavages or the exposure to solvent of the buried part of the protein during the denaturing and refolding (denaturing/refolding) processes. Here, we report the H/D exchange behaviors in hen egg-white lysozymes denatured under acidic conditions, basic conditions, and thermal conditions and then refolded in deuterated solvents, using crystallographic methods. The results indicate that the space containing the Trp28 side chain was hardly exposed to the solvent in acidic conditions, but exposed under basic or heated conditions. Moreover, the β-bridges between Tyr53 and Ile58 in strands β2 and β3, which are in a highly conserved region, show some tolerance to changes in pD. The results indicate that crystallographic method is one of the powerful tools to analyze the denaturing/refolding processes of proteins.

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Code Availability

The structure factors and coordinates have been deposited in the Worldwide Protein Data Bank under accession numbers 7FG8, 7FGU, and 7FGV for DLysCl, DLysOD, and DLyshc, respectively.

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Acknowledgements

We thank Dr. Andrey Kovalevsky and the staff members at the beamlines of IMAGINE, HFIR at the ORNL under the proposals IPTS-18574.1, 19637.1, 19782.1, and 20561.1; Dr. Alison Edwards and Dr. Ross Piltz for the KOALA, OPAL at the ANSTO under proposal P6087, for their help with neutron data collection; and Prof. Kunio Miki and his group at the Graduate School of Science, Kyoto University, for their kind help with X-ray data collection, biochemical experiments, and fruitful discussion.

Funding

This work was supported by the Photon and Quantum Basic Research Coordinated Development Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan (2013–2017 to A.K. and Y.M.), by the Research Development Program “Ishizue” of Kyoto University (2017 to A.K.), by the Future Development Funding Program of Kyoto University Research Coordination Alliance (2018 to A.K.), by The Towa Foundation for Food Science & Research (2018 to A.K.), and by The Kyoto University Foundation (2020 to A.K.).

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  1. Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kumatori, Sen-nan, Osaka, 590-0494, Japan

    Akiko Kita & Yukio Morimoto

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Kita, A., Morimoto, Y. Hydrogen/Deuterium Exchange Behavior During Denaturing/Refolding Processes Determined in Tetragonal Hen Egg-White Lysozyme Crystals. Mol Biotechnol 64, 590–597 (2022). https://doi.org/10.1007/s12033-022-00447-7

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