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Neutron Structure of the Enzyme DFPase

Nuclear density maps of DFPaseNuclear density for different regions of DFPase

Im the past year the number of entries in the Protein Data Base (PDB) has exceeded the number of 50.000. Most of these structure have been determined by X-ray crystallography, a smaller part by Nuclear Magnetic Resonance (NMR). Compared with this, the number of published neutron diffraction structures of proteins is tiny. Even though the first neutron structure of a protein was already solved in 1969 by Benno Schoenborn (sperm whale myoglobin, the same protein that John Kendrew used to determine the first X-ray structure of a protein ten years earlier) only about 20 proteins were characterized by neutron diffraction up to the present day.

Compared to X-ray diffraction the use of neutrons has a clear advantage. While hydrogen atoms are normally invisible in X-ray structures they become visible with neutrons. Knowing the location of hydrogen atoms means that protonation states of amino acid sidechains are known and that the orientation of water molecules can be determined. This is especially important for the understanding of enzyme machanisms. But why are there so few neutron structures? The reason is the very low flux of even modern neutron sources compared to the photon flux in modern synchrotrons used for X-ray crystallography. Therefore large crystals are required and long measurement time are necessary. Also the number of experimental stations dedicated for bio-macromolecules is limited. Currently only three sources allow the work on proteins (one in the USA, one in France and one in Japan).

The determination of the neutron diffraction structure of the enzyme diisopropyl fluorophosphatase (DFPase) was carried out at the spallation neutron source at Los Alamos National Laboratory (LANL) with one of the smallest crystals ever used for protein neutron crystallography. The volume of the crystal was 0.43 mm³. To record a complete data set about one month of beam time was required. The pulsed spallation source in Los Alamos also allowed the use of time-of-flight (TOF) techniques. The structure was deposited in the PDB under accession code 3BYC.

Rapid determination of hydrogen positions and protonation states of diisopropyl fluorophosphatase by joint neutron and X-ray diffraction refinement.
Blum MM, Mustyakimov M, Rüterjans H, Kehe K, Schoenborn BP, Langan P, Chen JC.
Proc Natl Acad Sci U S A. 2009 Jan 20;106(3):713-8.

Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms and macromolecular and solvent structure. Hydrogen atom positions can readily be determined by neutron diffraction, and as such, neutron diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement of neutron and X-ray diffraction data can lead to improved models compared with the use of neutron data alone and has now been incorporated into modern, maximum-likelihood based crystallographic refinement programs like CNS. Joint refinement has been applied to neutron and X-ray diffraction data collected on crystals of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of detoxifying organophosphorus nerve agents. Neutron omit maps reveal a number of important features pertaining to the mechanism of DFPase. Solvent molecule W33, coordinating the catalytic calcium, is a water molecule in a strained coordination environment, and not a hydroxide. The smallest Ca-O-H angle is 53°, well beyond the smallest angles previously observed. Residue Asp-229, is deprotonated, supporting a mechanism involving nucleophilic attack by Asp-229, and excluding water activation by the catalytic calcium. The extended network of hydrogen bonding interactions in the central water filled tunnel of DFPase is revealed, showing that internal solvent molecules form an important, integrated part of the overall structure.


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