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