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Enhanced hydrogen structure in enzyme intermediates using dynamic polarisation in neutron crystallography

Principal Supervisor: Peter Moody, Department of Molecular and Cell Biology

Co-supervisor: Emma Raven, Department of Chemistry

PhD project title: Enhanced hydrogen structure in enzyme intermediates using dynamic polarisation in neutron crystallography

University of Registration: University of Leicester

Project outline:

The efficient use of oxygen is essential for multi-cellular life. However, oxygen and its products can also but toxic. Heme in enzymes is key to many of the biological process involved in both its use and protection from it. We pioneered the combination of neutron crystallography with the cryo trapping of enzyme intermediates to gain insight in heme enzyme mechanism (Casadei et al., (2014) Science 345:193-197), and this proposal will allow us to combine our insights into the mechanism of heme enzymes with the development of new techniques to increase the scope and range of neutron crystallography.

Neutron crystallography uniquely allows direct observation of hydrogen atoms in protein structures; this is in contrast to X-ray crystallography where the low electron density of hydrogen and the short bond distance to neighbouring electron dense atoms together make reliable observation of hydrogen almost always impossible. However, hydrogen (1H) atoms have a negative scattering length and a very large incoherent scattering length. This incoherent scattering and the high proportion of hydrogen atoms in a protein structure means that the diffraction data has a very high background. The negative scattering length means that at moderate resolution we see cancellation effects. Exchangeable hydrogens can be substituted by using D2O, and the non-exchangeable hydrogens by growing the protein on deuterated carbon sources. Although H/D substitution helps a great deal, in practice large crystals are still needed and data collection is slow. Dynamic nuclear polarisation (DNP), whereby spin polarisation from electrons is transferred to nuclei, enables the incoherent scattering to be drastically reduced and the coherent scattering to be significantly increased (Zhao et al (2013) Physics Procedia 42: 39-45). This holds the promise of being able to improve the quality of neutron data from protein crystals and the insights available from detailed mapping of hydrogen positions. The application of the technique requires the introduction of a radical (via a reagent such as TEMPO) or paramagnetic centre. In the first instance we propose to exploit the paramagnetic heme iron in myoglobin for this. Myoglobin is robust and is been an established model system for neutron crystallography. We propose to look at the structure of an analogue of an intermediate of the heme peroxidase reaction (compound II) that can be formed in the crystal by reaction with hydrogen peroxide. The collection of the diffraction data using DNP will also require a sample environment at the neutron source capable of maintaining the magnetic fields and ultra-low temperatures required. We have established a collaboration with and Dr Leighton Coates (Lead Instrument Scientist, Macromolecular Neutron Diffractometer) at Oak Ridge National Laboratory (ORNL) and the University of Tennessee in the USA who is an experienced neutron scientist, committed to establishing and developing this technique for macromolecular structure determination. We expect some funding to become available through this collaboration to enable the student to spend some time at ORNL. During this time the student will take part in several cutting edge experiments utilizing the DNP technique on the operational neutron diffractometers MaNDi and IMAGINE.

 BBSRC Strategic Research Priority: Industrial Biotechnology and Bioenergy

Techniques that will be undertaken during the project:

  • Neutron Crystallography
  • X-ray crystallography
  • Nuclear magnetic resonance
  • Electron paramagnetic resonance
  • Single crystal spectroscopy
  • Enzymology
  • Dynamic nuclear polarisation

Contact: Dr Peter Moody, University of Leicester