An Effective Deuterium Exchange Method for Neutron Crystal Structure Analysis with Unfolding-Refolding Processes

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Neutron crystallography requires proteins with high deuterium content to minimize incoherent scattering.
• Fully deuterated proteins are costly and difficult to prepare.
• A simple and effective hydrogen/deuterium (H/D) exchange method is needed.

Purpose of the Study:

• To develop and evaluate a simple H/D exchange method for proteins.
• To assess the feasibility of using this method for neutron crystallography.
• To preserve the native structure of the protein after H/D exchange.

Main Methods:

• Hen egg-white lysozyme (HWL) underwent an unfolding-refolding process with H/D exchange.
• Deuteration levels were quantified using time-of-flight mass spectrometry.
• Refolded protein structures were analyzed using X-ray crystallography at 1.8 Å resolution.

Main Results:

• The H/D exchange method significantly increased deuterium content (over 20% of total hydrogen atoms) in HWL.
• X-ray structure analysis confirmed that the refolded HWL retained its native structure at atomic resolution.
• The deuteration was primarily concentrated in the protein's core.

Conclusions:

• The unfolding-refolding H/D exchange technique is an effective method for increasing protein deuteration.
• This method successfully preserves the native protein structure, making it suitable for neutron crystallography.
• It offers a cost-effective alternative to preparing fully deuterated proteins.