Research Article: Cryoprotectant-free high-pressure cooling and dynamic nuclear polarization for more sensitive detection of hydrogen in neutron protein crystallography

Date Published: August 01, 2018

Publisher: International Union of Crystallography

Author(s): Ichiro Tanaka, Naoya Komatsuzaki, Wen-Xue Yue, Toshiyuki Chatake, Katsuhiro Kusaka, Nobuo Niimura, Daisuke Miura, Takahiro Iwata, Yoshiyuki Miyachi, Genki Nukazuka, Hiroki Matsuda.


To improve the sensitivity of hydrogen detection using neutrons, high-pressure cooling of a relatively large protein single crystal and proton polarization of a protein polycrystalline state have been demonstrated as preliminary experiments.

Partial Text

It is easier to detect hydrogen in macromolecules using neutron protein crystallography (NPC) than using X-ray protein crystallography (XPC). However, it may be difficult to detect H atoms involved in the chemical reactions catalyzed by enzymes using conventional neutron methods because they have low occupancies (Niimura & Bau, 2008 ▸; Blakeley et al., 2008 ▸; Blakeley, 2009 ▸; Niimura & Podjarny, 2011 ▸; Niimura et al., 2016 ▸). The nuclear polarization technique has been explored over a long period of time in order to attempt to overcome technical difficulties related to neutron scattering. The dynamic nuclear polarization (DNP) method can align the direction of nuclear spins by transferring the spin polarization from electrons to the nuclei so that the neutron scattering length of each element varies (Abragam & Goldman, 1978 ▸). Application of DNP to the field of neutron protein scattering has been attempted as an alternative method to deuterium substitution (Stuhrmann et al., 1986 ▸; Knop et al., 1992 ▸; Zhao et al., 1995 ▸; Stuhrmann, 2004 ▸). At present, with the emergence of next-generation neutron sources, DNP of hydrogen in proteins is expected to improve the sensitivity of hydrogen detection by about eight times compared with normal NPC and to reduce the background from hydrogen incoherent cross-sections (Pierce et al., 2010 ▸; Niimura & Podjarny, 2011 ▸; Zhao et al., 2013 ▸, 2016 ▸; Tanaka et al., 2013 ▸). There have been some reports of proton polarization in neutron diffraction and scattering experiments (Zimmer et al., 2016 ▸; Piegsa et al., 2013 ▸). Applications to NPC, however, have not yet been performed. Several technical difficulties need to be overcome in order to realize the DNP method in NPC: cooling a large protein single crystal and obtaining a higher proton-polarization rate for a protein sample doped with a suitable concentration of a radical molecule at low temperature (∼1 K) in a high magnetic field (∼3 T).

As preliminary experiments for improving the sensitivity of hydrogen detection using neutrons, high-pressure cooling and proton polarization of protein crystals have successfully been demonstrated.(i) A relatively large protein single crystal could be cooled without any cryoprotectants at 200 MPa. The crystal diffracted X-rays to high resolution and the structures were almost the same as those obtained with normal cooling according to the r.m.s.d.s.(ii) ESR measurements on a single protein crystal showed a good proportionality between the radical concentration used in protein crystal growth and the number of radicals per protein molecule. This information is useful for finding the best concentration of radicals in a protein single crystal sample for DNP.(iii) The first DNP experiment with TEMPOL in the protein polycrystalline state was conducted and the maximum polarization rate obtained was 22.3 ± 0.7%.




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