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X-ray structure of perdeuterated DFPase – perdeuteration of enzymes for neutron diffraction

Plot der RMSD Werte für C-alpha Kohlenstoffatome zwischen d-DFPase und h-DFPase

Plot of C-α RMSD values between d-DFPase and h-DFPase

Protein structures solved by neutron diffraction have the significant advantage compared to X-ray structures that hydrogen atoms are clearly observable in the nuclear density maps. This allows the determination of protonation states in amino acid side-chains and the orientation of solvent molecules (especially water) in space. This is of special importance for the elucidation of enzyme mechanisms. The disadvantages of neutron diffraction with proteins exist in the small number of powerful neutron sources and dedicated instruments worldwide. Also – neutron flux even at the most powerful sources is small compared to photon flux at X-ray sources. Therefore large protein crystals are required and data collection times can easily be in the region of several weeks. Another problem is the signal to noise ratio. Hydrogen atoms in the crystal are major contributors to this problems because the large incoherent scattering cross section of hydrogen. High values for this incoherent scattering cross section lead to diffuse scattering and negatively influence the signal to noise ratio. Deuterium on the other hand displays a significantly smaller value (2.05 b for deuterium compared with 80.27 b for normal hydrogen; 1 b = 100 fm²). To overcome this problem, crystals grown with hydrogenous protein are normally soaked with deuterated mother liquor (or brought in contact via gas diffusion).

Cover of Acta F with DFPase

Cover of Acta F with DFPase

Labile hydrogens (e.g. those of water in the solvent of in acidic or basic functional groups in the protein) are exchanged with deuterium. Non-labile hydrogens like those in aliphatic or aromatic C-H bonds are no exchanged. Therefore a significant number of hydrogen atoms remain in the protein. Such a partially exchanged crystal was used for the already published neutron structure of DFPase.

To achieve full deuteration of the protein it has to be grown in fully deuterated media. We now report the X-ray structure of fully deuterated DFPase in a new publication in the journal Acta Cryst. F. The structure (solved at room temperature at a resolution of 2.1 Å) shows that full deuteration leads to practically no changes in the protein structure. But even though a very large crystal of d-DFPase was grown (> 2mm³) it did not diffract neutrons beyond very low resolution. An explanation for this unexpected result can be found either in the differences in data aquisition (cross section of the neutron beam compared to the X-ray beam used) or can be based on crystallographic parameters. The scattering characteristics of successful neutron experiments and associated X-ray data are presented in tabulated form and can serve as a guidance for future neutron experiments.

X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase): Perdeuteration of proteins for neutron diffraction.
Blum MM, Tomanicek S, John H, Hanson L, Rüterjans H, Schoenborn BP, Langan P, Chen JC.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66(4):379-385.

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The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase) is reported at 2.1 Å resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallographic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 Å resolution appears to be a strong predictor of successful neutron structures.


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