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.

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).

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.

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.

https://www.sciencedirect.com/science/article/pii/S0003986118300535?via%3Dihub

After applying lactate dehydrogenase catalysis to the synthesis of perdeuterated enantiopure lactic acid, the ESS deuteration lab is now extending its use of enzyme catalysis to the lipase class of enzymes. The natural function of these enzymes is to hydrolyse ester bonds of triglycerides, releasing free fatty acids, under aqueous conditions. Lipases are also remarkably stable in organic solvents, and so can be encouraged to perform the reverse esterification reaction under water-free or water-limited conditions (Scheme 1).

Lipases are already used extensively in synthetic chemistry, so much so that many of them are commercially available in immobilised form. The ESS lab has recently procured a rotating bed reactor (RBR – Figure 1), and some lipases immobilised on various solid supports, contained within cartridges designed to be used in combination with the RBR. This equipment should allow the use of lipases to perform efficient synthetic transformations of deuterated molecules using immobilised enzymes.

Initial work has begun with a simple, unlabelled esterification system (Scheme 2). The reaction can easily be monitored by GC-FID, allowing for analysis of reaction progress and kinetics (Figure 2).

Work will soon continue with more complex, deuterium-labelled systems to produce deuterated materials of use in neutron scattering experiments.

J-PARC (Japan Proton Accelerator Research Complex) last month held a workshop entitled “Deuterated Materials Enhancing Neutron Science for Structure Function Applications”, at the Ibaraki Quantum Beam Research Centre, Tokai, Japan. There were over 60 participants in the workshop, which was held over two days. J‑PARC have recently established on-site laboratories dedicated to chemical and biological deuteration and the aim of the workshop was to discuss how to activate deuteration science at J‑PARC.

Three plenary speakers outlined the current status of other deuteration laboratories around the world. Dr Tamim Darwish and Dr Anthony Duff from ANSTO described the chemical and biological deuteration laboratories at the National Deuteration Facility, ANSTO, which have been functioning well as user laboratories for several years. Dr Anna Leung presented the research at the newly-established DEULAB at ESS, and also acted as a representative for the other members of the DEUNET (the laboratories at ISIS, ILL and FZJ). Professor Sajiki from Gifu Pharmaceutical University provided the fourth plenary session. Professor Sajiki has worked in the field of deuteration science for many years, and presented some of his recent results in the field.

Ten invited talks provided an excellent overview of the current research involving deuterated materials in Japan, and initiated excellent discussion about the future of deuterated materials research, and where more effort should be focussed to produce high-impact research. The meeting concluded that a deuteration community ought to be organised in Japan to leverage the advantages of collaborative research. Furthermore, this community ought to continue to interact with deuteration laboratories across the world. In addition to the existing collaboration between J‑PARC and the chemical deuteration laboratory at ANSTO, J‑PARC has joined the European chemical deuteration network (DEUNET) as an observer member, a development which is certain to benefit both deuteration communities and their users.

J-PARC (Japan Proton Accelerator Research Complex) is a multidisciplinary research facility with a series of proton accelerators producing neutron, pion, kaon and neutrino beams. This infrastructure is used to investigate research questions in areas including nuclear and particle physics and materials and life sciences. J‑PARC has recently built biology and chemistry laboratories in which research will focus on producing deuterated molecules for use in neutron experiments, and this month held a workshop entitled “Deuterated Materials Enhancing Neutron Science for Structure Function Applications”.

Japan has a strong reputation in the field of deuteration science, most notably in the form of Professor Sajiki at Gifu Pharmaceutical University. In Japan, a deuteration science community is beginning to organise around existing expertise, and new research which will be done at the J‑PARC deuteration laboratories. The chemical deuteration laboratory at J‑PARC has a strong collaborative project with the chemical deuteration laboratory at the National Deuteration Facility, ANSTO (AUS), with exciting developments being made in the area of highly and selectively deuterated ionic liquids.

The Deuteration Network members welcome J‑PARC to the DEUNET. We are certain this will bring benefits to all of the members and their respective user communities, and we welcome the exciting collaborations we anticipate in the near future.

The chemical deuteration laboratory at ESS (DEULAB) is working to establish innovative methods of producing complex perdeuterated molecules with exciting applications in neutron scattering.

The first method focuses on the production of perdeuterated chiral molecules. Chiral molecules exist in two different forms (enantiomers) which are mirror images of each other but otherwise identical. The synthesis of a chiral molecule as a single enantiomer, without its mirror image, is challenging, and the inclusion of isotopic labelling imposes even more requirements upon equipment, methods and synthetic design. This is one reason why the number of perdeuterated chiral molecules available to the neutron scattering community is so limited.

Our method uses enzyme catalysts, which, due to the intricate design of their active site, often have full specificity for one enantiomer only, and can transform relatively economical, achiral precursors into valuable chiral molecules. Using deuterated analogues of  the precursors allows us to use this process to produce deuterium-labelled chiral molecules. Our first target molecule was lactic acid, which exists as both D-lactic acid, and L‑lactic acid (Figure 1). Lactic acid is best known as the molecule produced in the body during exercise, but it is also of interest in the fields of material science and biomedical technology as the monomer for poly(lactic acid) polymers which are renewable, biodegradable and biocompatible.

Figure 1. The two enantiomers of lactic acid.

The lactate dehydrogenase enzyme is responsible for the production of lactic acid from pyruvate during normal metabolism and exercise, and normally it exists in only the L-form producing only L-lactic acid. However, D‑lactate dehydrogenase, which produces and acts exclusively on the other enantiomer D-lactic acid, has also been expressed recombinantly in bacterial hosts. We first used D‑lactate dehydrogenase to produce perdeuterated D-lactic acid-d₄. In addition to the enzyme, we used two deuterated reagents, sodium pyruvate-d_3, which we can easily synthesise in our lab and sodium formate-d₁, which is commercially available. The other components of the catalytic system, including a buffer, a coenzyme, an acid and water as solvent – were all standard, unlabelled reagents. We isolated the product as D-lactic acid-d₄ and analysed it using mass spectrometry to determine the level of deuterium incorporation (Figure 2). Pleasingly, we could see that the molecular weight of the product was four atomic mass units higher than for unlabelled lactic acid (one atomic mass unit per deuterium atom).

Figure 2. Top: mass spectrum of unlabelled lactic acid (89.0 amu). Bottom: mass spectrum of D-lactic acid-d₄; shifted by amu (93.0 amu).

This process is equally applicable to producing perdeuterated L-lactic acid-d₄, by using L-lactate dehydrogenase as the catalyst. Both samples will be used by our Deuteration Network (DEUNET) partners at the Jülich Research Centre (FZJ) in Germany to synthesise tailored poly(lactic acid) samples, which will be analysed by neutron scattering to correlate the arrangement of the chiral monomers with the structure and physical properties of the polymers.

The number of similar enzymes which are commercially available, and the substrates they accept, suggests that this method will have broad applicability in the synthesis of chiral deuterated molecules, and so, at ESS, we are developing this enzyme-catalysis to produce more novel perdeuterated chiral molecules which are presently unavailable.

From Monday 15^th-Wednesday 17^th May, the Deuteration for Neutron Scattering – DEUNET Workshop was held in Oxford, England, organised jointly by the STFC Deuteration Facility and the European Deuteration Network (DEUNET).

Prominent researchers working in the fields of lipids and membranes, energy materials, surfactants, polymers, protein structure and function, colloids, and other small molecules, presented their research which has exploited the power of the tandem techniques of deuterium-labelling and neutron scattering. The programme demonstrated the impact that deuterium labelling has on neutron science, and the ways in which this leads to high-impact science. Each of the facilities involved in the European Deuteration Network presented the current capabilities of their laboratories, in addition to projects and development being undertaken. It was very enjoyable to host delegates from other facilities around the world who work in the field of deuteration, such as J-PARC (Japan) and ORNL (USA), and to hear about the work undertaken in their laboratories.

There was a great deal of opportunity for delegates to interact outside of the presentations, during discussion sessions, a poster session, and within meal and coffee breaks. An advisory board was appointed for the DEUNET, comprised of Trevor Forsyth (ILL), Jian Lu (University of Manchester), Thomas Hellweg (Bielefeld University) and Peter Holden (ANSTO). We trust that they will succeed in providing the network with invaluable advice and insight.

We hope to hold the next STFC – DEUNET Deuteration for Neutron Scattering workshop in late 2018. Further information will be provided at Deuteration.net as it becomes available.

Thanks to all those involved in the workshop for a productive and inspiring meeting!

The delegates of the Deuteration for Neutron Scattering – DEUNET Workshop held in Oxford, 15-17^th May 2017.