Principal Supervisor: Dr Peter Moody, Department of Molecular and Cell Biology
Co-supervisor: Dr Emma Raven, Department of Chemistry
PhD project title: Neutron crystallography to study heme enzyme intermediates
University of Registration: University of Leicester
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), Kwon et al., (2016) Nature Communications 7, 13445) 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. Some experimental work and data collection will take place with our collaborator Dr Leighton Coates at Oak Ridge National Laboratory (ORNL) the USA. We expect funding to become available for an extended (up to two years) placement at ONRL.
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 unpaired electrons. ONRL are developing the instrumentation (and sample environment at the neutron source capable of maintaining the magnetic fields and ultra-low temperatures required) on the reactor (HiFER) IMAGINE beam line using the radical approach. We propose to extend the range of the techniques by exploiting 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. Our established collaboration with Dr Coates (who is the Lead Instrument Scientist, of the Macromolecular Neutron Diffractometer (MaNDI) on the spallation neutron source at ORNL) will provide access to the instrumentation, facilities and expertise at ONRL. The project will involve the student taking part in cutting edge experiments utilizing the latest advances in macromolecular neutron crystallography.
BBSRC Strategic Research Priority: Molecules Cells and Systems
Techniques that will be undertaken during the project:
- Neutron Crystallography
- X-ray crystallography
- Nuclear magnetic resonance
- Electron paramagnetic resonance
- Single crystal spectroscopy
- Dynamic nuclear polarisation