Tasks

Deuterated biomass: optimisation. A large number biological neutron studies rely on the
biosynthesis of deuterated biological macromolecules using deuterated carbon sources. The cost of
these often limits experiments severely. In this task protocols will be developed for the optimisationof algal biomass production. Since algae grow photosynthetically, relying only on D2O medium, CO2, and light, they are an ideal candidate for cheaper production of deuterated components1. Major downstream applications in neutron scattering are obvious for these in vivo products. This task will link with 18.7, focused on chemical synthesis of small biomolecules.

Most proteins used in neutron scattering studies are produced in bacteria such as E. coli. This type of protein production has had a huge impact on studies of biological systems using a wide variety of techniques. In many cases the use of E. coli is limited by folding/post translational modification problems2. Some proteins have to be produced in lower eukaryotic expression systems. However there are difficulties in adapting such cells to growth in deuterated media. Here we will develop methods whereby labelled proteins can be produced (intracellularly or exported) in yeast. The successful development of such systems will open up completely new areas for neutron-based biomolecular studies.

There is a huge requirement for proteins where extended regions/domains/subunits are selectively deuterated. Protein splicing approaches do exist for this type of segmental labelling, but methods for 2H labelling have not been developed. Development the method for deuteration will allow contrast matching studies of the type described by Callow et al (2007)3 to be carried on a far wider range of sample systems. Two general strategies have been proposed for segmental isotopic labelling: transprotein splicing activity of split inteins4, and native chemical ligation. We will evaluate and compare both schemes using a two-domain test protein. The most efficient method will then be used to establish a reliable protocol for neutron scattering. Complementary NMR studies will also be carried out to evaluate the efficacy of the labelling and also to pursue the synergy in combined SANS and NMR solution approaches.

The high cost of deuterated carbon sources for the expression of D-proteins in E.coli is still a
stumbling block for the application of neutrons in biology. In this task, methods will be developed for the production of deuterated glycerol by algae under salinic stress. We will develop a novel protocol based around “milking” the algae of the glycerol produced, dramatically reducing the cost since the same biomass and D2O can be used repeatedly. Approaches will be developed using Dunaliella salina. This strain accumulates up to 6M glycerol in the cell and this task will focus on efficient release of the glycerol into the media. Methods to be tested will include high temperature treatment, cell immobilisation and mild sonication.

Membrane proteins perform a wide range of essential cellular functions and play key role in (for example) transportation, energy management, signal transduction, photosynthesis. They are also implicated in a number of genetic diseases and have considerable therapeutic importance (70% of drug targets). This sub task will be focused on the development of methods to optimise deuteration of membrane proteins. Model membrane proteins will be identified and bacterial expression systems in high cell density cultures will be used for deuteration. The deuterated membrane proteins will be used to reconstitute membrane systems with hydrogenated lipids for neutron studies and to test new classes of surfactants for their capacity to stabilise functional assemblies.

This work package will build on the capability within the recently funded Small Molecule
Deuteration Facility (funded by STFC, based at Oxford). One of the major limitations for the
application of neutrons to the study of biological systems is the availability of deuterated lipids. Membrane biology is a particularly important area and a new range of powerful experiments could be carried out with the availability of selectively labelled lipids11. In this task, the highest priority is the provision of unsaturated perdeuterated lipids with a range of head groups, requiring the development of an optimised route for the production of oleic acid and conversion to the target lipids.