title: An Effective Deuterium Exchange Method for Neutron Crystal Structure Analysis with Unfolding-Refolding Processes.
abstract: A method of hydrogen/deuterium (H/D) exchange with an unfolding-refolding process has been applied to hen egg-white lysozyme (HWL), and accurate evaluation of its deuteration was carried out by time-of-flight mass spectroscopy. Neutron crystallography requires a suitable crystal with enough deuterium exchanged in the protein to decrease incoherent scattering from hydrogens. It is very expensive to prepare a fully deuterated protein, and therefore a simple H/D exchange technique is desirable for this purpose. Acid or base addition to protein solutions with heating effectively increased the number of deuterium up to more than 20 % of that of all hydrogen atoms, and refolded structures were determined by X-ray structure analysis at 1.8 Å resolution. Refolded HWL had increased deuterium content in its protein core and its native structure, determined at atomic resolution, was fully preserved.
authors.affiliation: Division of the Quantum Beam Material Science, Research Reactor Institute, Kyoto University, Kumatori, Osaka, 590-0494, Japan. kita@rri.kyoto-u.ac.jp.
authors.forename: Akiko
authors.initials: A
authors.lastname: Kita
authors.affiliation: Division of the Quantum Beam Material Science, Research Reactor Institute, Kyoto University, Kumatori, Osaka, 590-0494, Japan. morimoto@rri.kyoto-u.ac.jp.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Morimoto
mesh_terms: D002942:Circular Dichroism; D018360:Crystallography, X-Ray / Q000379:methods*; D003903:Deuterium / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D009113:Muramidase / Q000737:chemistry; D058849:Protein Refolding; D058767:Protein Unfolding
chemical_list: D003903:Deuterium; C421864:hen egg lysozyme; D009113:Muramidase
keywords: H/D exchange; High-resolution X-ray analysis; Protein unfolding–refolding; TOF mass spectroscopy