Journal
MOLECULAR BIOTECHNOLOGY
Volume 58, Issue 2, Pages 130-136Publisher
HUMANA PRESS INC
DOI: 10.1007/s12033-015-9908-8
Keywords
H/D exchange; Protein unfolding-refolding; High-resolution X-ray analysis; TOF mass spectroscopy
Funding
- Photon and Quantum Basic Research Coordinated Development Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan
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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 resolution. Refolded HWL had increased deuterium content in its protein core and its native structure, determined at atomic resolution, was fully preserved.
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