Fundamental studies for the proton polarization technique in neutron protein crystallography

Ichiro Tanaka,Toshiyuki Chatake,Katsuhiro Kusaka,Nobuo Niimura

doi:10.1107/s0909049513020815

Ichiro Tanaka, Toshiyuki Chatake + Show 2 more

Open Access

https://doi.org/10.1107/s0909049513020815

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Abstract

The isotope effect in conventional neutron protein crystallography (NPC) can be eliminated by the proton polarization technique (ppt). Furthermore, the ppt can improve detection sensitivity of hydrogen (relative neutron scattering length of hydrogen) by approximately eight times in comparison with conventional NPC. Several technical difficulties, however, should be overcome in order to perform the ppt. In this paper, two fundamental studies to realise ppt are presented: preliminary trials using high-pressure flash freezing has shown the advantage of making bulk water amorphous without destroying the single crystal; and X-ray diffraction and liquid-chromatography/mass-spectrometry analyses of standard proteins after introducing radical molecules into protein crystals have shown that radical molecules could be distributed non-specifically around proteins, which is essential for better proton polarization.

Highlights

Hydrogen, protonation and hydration play important roles in various biological functions at the atomic level
Lysozyme single crystals grown with the precipitant containing PEG, the dimensions of which were a crystal edge size of around 0.5 mm, were successfully frozen transparently
As larger crystals are necessary in an actual neutron protein crystallography (NPC) experiment, the freezing of relatively large biological crystals requires a more rapid cooling technique, pressures higher than 160 MPa, and more suitable cryoprotectants for a reliable cooling method

Summary

Introduction

Protonation and hydration play important roles in various biological functions at the atomic level. Neutron protein crystallography (NPC) is one of the most powerful techniques for investigating these phenomena concerning hydrogen (Tsyba & Bau, 2002; Niimura & Bau, 2008; Tanaka et al, 2010). There always remains the question of whether threedimensional structures in deuterated and undeuterated (native) proteins are identical or not, because living cells cannot survive in the heavy water environment for long; in other words, the isotope effect. This effect in conventional NPC can be eliminated by the proton polarization technique (ppt) (Niimura & Podjarny, 2011).

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