The DEUNET is growing! Our most recent meeting was held in Lund last week and included several new members. The Lund Protein Production Platform have joined the DEUNET, and biological deuteration and crystallisation at ESS has joined chemical deuteration in the network. Larodan AB also joined their first DEUNET meeting, and we were very fortunate to have Professor Hironao Sajiki from Gifu Pharmaceutical University, Japan, present some of his recent work in the field of deuteration chemistry.
The member labs provided updates and several possible collaborative projects were identified. We are, as always, grateful to the DEUNET advisory panel for travelling to the meeting and providing valuable advice. The DEUNET advisory panel consists of both deuteration facility members (Peter Holden from ANSTO, and Trevor Forsyth from ILL), and neutron users (Karen Edler, Jian Lu and Thomas Hellweg).
The ESS DEMAX platform recently published its first call for proposals for deuteration and crystallisation support. Nineteen proposals were received, covering chemical deuteration, biological deuteration and crystallisation. The review process is underway and results will be communicated by the end of May. Thanks to all those neutron experimenters who submitted proposals!
BrightnESS² is a three-year, EU-funded project within the European Commission’s Horizon 2020 Research and Innovation programme which focuses on the long-term sustainability of ESS and neutron scattering in Europe, and to further strengthen the network of facilities for research using neutrons. A total of 15 institutes and universities from Europe and South Africa are participants within the project.
As part of BrightnESS² the Deuteration Facility at the SFTC (UK) and the ESS Chemical Deuteration Laboratory (SE) are collaborating on the synthesis of structured deuterated phospholipids. At the moment, a limited number of deuterated, mixed acyl phospholipids (Figure 1) are available, and those that are available and prohibitively expensive for many neutron users and experiments.
Mixed acyl phosphocholine lipids, where R1 and R2 are different acyl tails.
The STFC Deuteration Laboratory has a wealth of experience in the synthesis of homo-acyl phospholipids and an active user community which continuously requests new phospholipid analogues for neutron experiments. The ESS Chemical Deuteration Laboratory is researching the application of enzymatic catalysis to the synthesis of deuterated molecules. Together, the facilities are working together to establish a new, modular method for the synthesis of tail-deuterated, mixed-acyl phospholipids, using enzymes to allow the regiospecific substitution of tails at the 1- and 2-positions. The modularity of the approach should mean that a large number of molecules can be accessed. The first target molecules are 1-palmitoyl-d31-2-oleoyl-d33-sn-3-phosphocholine (POPC-d64, Figure 2a) and 1-palmitoyl-d31-2-(9-oxononanoyl)-sn-3-phosphocholine (PoxnoPC-d31, Figure 2b).
The Deuteration and Macromolecular Crystallisation (DEMAX) Platform at ESS has opened its first call for proposals. DEMAX offers three pillars of support: biological deuteration (e.g. cell paste, soluble proteins, lipids, membranes), chemical deuteration (e.g. small organic molecules, surfactants, phospholipids) and crystallisation (biological macromolecules e.g. proteins).
At this time, DEMAX offers the following kinds of deuterated materials and services:
Total yeast-derived lipids
Tail-deuterated lipids
Saturated fatty acids/alcohols/halides/thiols
Oleic acid
Surfactants (e.g. sugar, amino acid)
Lactic acid
Bacterial cell pastes
Recombinant proteins
Support for large protein crystal growth (incl. sitting drop vapour diffusion, dialysis, batch, optimisation, targeted screening, testing with X-rays)
For this call, access is not limited to ESS member countries, and is free of charge. The deadline for proposal submissions is 5th April 2019. Find more information here: https://europeanspallationsource.se/node/244384
Questions? Contact the DEMAX team at demax@esss.se
The ESS Deuteration and Macromolecular Crystallisation (DEMAX) Platform will publish its first call for service requests for deuterated materials and support for large crystal growth for neutron experiments. The call be open from February – April 2019 – watch this space for more information!
The ESS DEULAB installed its first Parr reactor during the summer. The Parr reactor is capable of operating under conditions of high temperature (up to 350 °C) and high pressure (up to 131 bar) and is used in deuteration laboratories for hydrothermal, metal-catalysed H/D exchange reactions.
The first reaction performed in the DEULAB using the Parr reactor was the deuteration of lauric acid:
Under these conditions, lauric acid with 89% deuteration of the non-exchangeable hydrogen atoms was obtained. The deuteration incorporation was determined by analysis of the isotopologue ratios in the mass spectrum:
This process can be repeated to produce very highly deuterated lauric acid (>98% deuteration of the non-exchangeable hydrogen atoms). It is applicable to other saturated fatty acids and will facilitate the synthesis of many classes of deuterated molecules with fatty acid synthons, such as lipids and surfactants, at the ESS DEULAB.
A team from Lund University, Karolinska Institute, and the European Spallation Source have published an optimized approach to high-yield protein deuteration at low cost, for neutron protein crystallography.
RELEVANCE
In general, neutron scattering experiments in life sciences require deuterated bio-reagents. In particular for neutron protein crystallography (NPX) studies there is a need for deuterated proteins in sufficient quantities to allow for crystallization and scale-up to obtain large (0.5 – 1.0 mm3) single crystals for neutron data collection (Figure 1). Our results demonstrate the progress in our abilities to produce deuterated proteins at an improved cost-to-yield ratio.
Figure 1. Photograph of a large, single crystal of human carbonic anhydrase.
BACKGROUND
Deuteration means to exchange Hydrogen (H) atoms with Deuterium (D) atoms. This is a crucial technique that allows us to benefit from the stronger neutron scattering power and lower background from D compared to H in NPX. For the production of recombinant proteins, deuteration is done in the production hosts, e.g. bacteria. While the benefits are clear, there are many drawbacks to deuteration, including high costs, toxic effects of D towards the production hosts resulting in low yield of biomass, and possibly changes in the biophysical properties of the recombinant protein, altogether, making it technically difficult to produce and crystallize deuterated proteins.
In our current study “Deuteration of human carbonic anhydrase for neutron crystallography: cell culture media, protein thermostability, and crystallization behavior” we address the challenges of expressing proteins in bacteria under deuterated conditions. Technical difficulties, such as cellular toxicity that results in reduced yields of recombinant protein and the high cost of deuterated growth media for the production, makes it challenging to produce enough deuterated protein for NPX studies. Furthermore, there are effects of full deuteration on protein stability and solubility. In almost all reported cases deuteration causes a reduction in thermal stability and solubility and consistently leads to smaller crystal sizes, compared to what is possible with the hydrogenous versions.
Our study was designed to address some of the technical difficulties of protein deuteration in vivo in bacteria and to study the biophysical effects with regards to thermal stability and crystallization behaviour of the products. We started with a survey of the literature and the global protein structure database (https://www.rcsb.org/) to reveal the most common approaches used for deuteration of proteins in NPX. Our goal was to prepare partially deuterated samples, in a short time with the best possible yield for the cost.
We combined further optimized approaches that still gave very good deuterium incorporation with optimal protein yields, but also significantly reduced the time and cost for producing the proteins compared to established protocols. After producing and isolating the deuterated proteins, we measured the D incorporation with mass spectrometry and tested thermal unfolding as a function of pH to characterize protein stability and solubility. Finally we tried to grow crystals of deuterated and non-deuterated proteins side-by-side to assess the impact of deuteration on crystallization behavior.
RESULTS
In this study we optimized the deuterated production of 3 different versions of human carbonic anhydrases. Resulting from this are a cost effective cell culture medium and optimization of the cell culturing and expression protocols. Our analysis showed that the deuterated proteins were on average 1 – 4 °C less stable than the hydrogenous versions and this effect was strongly pH-dependent. Interestingly, deuterated proteins dissolved in D2O were more stable than deuterated proteins in H2O. However our partial deuteration strategy did not seem to affect protein solubility in the concentrations necessary for crystallization. Comparing crystallization trials of deuterated and non-deuterated proteins at the same pH indicated that deuteration had significant effects on crystallizability (Figure 2).
Figure 2. Side-by-side comparison of non-deuterated and deuterated protein crystallization behavior. Some proteins did not produce any crystals in the deuterated form.
In summary, our protein yield was ~2-fold lower than in non-deuterated conditions, but still producing tens of milligrams of pure protein, yields sufficient for NPX. We managed to get ~65-80% D incorporation, also sufficient for NPX applications, at a cost that was reduced by ~4 fold compared to previously reported protocols. We will adopt these methods to prepare deuterated samples for future neutron experiments.
FUTURE
These results are exciting and will help us be more efficient in deuterated protein production. However, there is still some work to be done. In particular, production of deuterated protein is still ~120 fold more expensive as non-deuterated. So can we further optimize costs?
Also, as our studies indicate, there is a pH-dependent effect of deuteration on crystallization behavior and we will aim to optimize and find ways to adapt our conditions to maximize our chances of growing large crystals of deuterated proteins.
WHO?
The experimental team involved several scientists from Lund University, Karolinska Institute, and the European Spallation Source (DEMAX platform): Katarina Koruza, Benedicte Lafumat, Ákos Végvári, Wolfgang Knecht, and Zoe Fisher. The work was done as a collaboration between Lund University (Lund Protein Production Platform, LP3 www.lu.se/lp3) and the European Spallation Source (ESS) DEMAX platform.
Figure 3. Katarina Koruza inspecting crystallization plates under the microscope.
Funding for this work was received by the Royal Physiographic Society of Lund, Interreg/MAX4ESSFUN, The Crafoord Foundation, BioCARE and EU’s Horizon 2020 research and innovation program (SINE2020).
REFERENCE: Koruza K, Lafumat B, Végvári Á, Knecht W, Fisher SZ (2018) “Deuteration of human carbonic anhydrase for neutron crystallography: Cell culture media, protein thermostability, and crystallization behavior”, Arch Biochem Biophys. 645, p.26-33.
