A joint EMBL/ILL/IBS Deuteration and Isotope laboratory has been established as part of the Partnership for Structural Biology (PSB).
Its purpose is to develop tools and expertise needed to produce deuterated materials for neutron scattering experiments and NMR as well as improved systems and technologies for the production and analysis of deuterium-labelled biological macromolecules.
Neutron protein crystallography can provide a powerful complement to X-ray crystallography by enabling key hydrogen atoms to be located in biological structures that cannot be seen by X-ray analysis alone. The neutron Laue diffractometer LADI, run jointly by EMBL and ILL at the ILL high flux reactor in Grenoble, is a dedicated facility for neutron protein crystallography at high-resolution (1.5 Å) and provides 10-100 fold gains in efficiency compared with conventional neutron diffractometers. The availability of fully deuterated protein eliminates the hydrogen incoherent scattering contribution to the background and brings further ~10-fold improvements in the signal to noise ratios.
The production of D-labelled macromolecules is done using bacterial expression systems. Growth on deuterated media is slow and produces lower yields of biomass and recombinant proteins. The molecular response mechanism(s) underlying bacterial adaptation to deuterium are unknown. In order to understand and improve the adaptation process, we investigate the deuterium effect on E. coli in a comparative proteomic approach using 2D gel electrophoresis. Alternatively, an E. coli expression library is used to identify key proteins that could trigger more efficient bacterial adaptation.
A major hurdle to neutron protein crystallography is that unusually large crystals (~1mm3) are required to compensate for the weak flux of available neutron beams. A method and a device for the promotion of crystal growth by keeping the growth solution metastable during the growth process have been developed. This works by regulating the temperature of the growth solution using control parameters determined in situ during the growth process.
Whilst the X-ray structures of perdeuterated and hydrogenated proteins are essentially indistinguishable to near atomic resolutions, the subtle differences in fixed-point properties of D2O and H2O affect protein solubility, intermolecular interactions and association between H/D macromolecules in a small but significant way. We are characterising the effects of D2O and deuteration on the physico- chemical properties of bio-macromolecules in order to help optimise the crystallisation of deuterium-labelled biological macromolecules. Solubility measurements with several diverse H/D protein systems were measured and indicate that the replacement of H2O by D2O in general decreases the solubility due to an increase in the attractive intermolecular interactions in solution.