Production of Perdeuterated Proteins in Escherichia coli for Structural Studies by Neutron Crystallography

Sammanfattning: Neutron macromolecular crystallography (NMX) is a complementary technique to X-ray crystallography leveraging neutron scattering properties of the hydrogen isotope deuterium (2H). Production of deuterium labelled molecules together with large crystal requirements for neutron crystallography are few of several bottlenecks limiting implementation of this technique for a wider range of proteins. Perdeuterated protein production is carried out as conventional recombinant protein production with the differences that the production host is grown in a medium with deuterium enriched water (heavy water) and a deuterated carbon source. It is reported that Escherichia coli and other microorganisms, in heavy water-based growth medium experience reduced growth rate and biomass yield. The aim of this work was to study and improve growth of E. coli in deuterated media for production of perdeuterated proteins for NMX and in parallel use NMX to study the reaction mechanism of the enzyme triose-phosphate isomerase (TIM). TIM is a key enzyme in glycolysis and it catalyzes the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate via proton transfer. Thus, neutron crystallography will provide additional information on the protonation state of the active site.Paper I and Paper II describes two different approaches to obtain strains which grow faster in deuterated growth media. An in vivo mutagenesis approach was used in Paper I and in Paper II long term evolutionary adaptation was used. Both approaches were successful in obtaining strains with improved growth properties. However, the analyzed strains had different mutations and showed either a general improvement of growth in minimal media or a specific improvement in deuterated medium. The obtained strains were engineered for recombinant protein production and shown to be suitable for production of perdeuterated proteins.Perdeuteration of TIM and an atomic model based on joint X-ray/neutron diffraction data is presented in Papers III and IV. Paper III reports perdeuteration of this protein using a strain obtained in Paper I, as well as a procedure to produce large volume crystals. This study emphasizes that larger crystals gives data with better statistics. Paper IV reports structure of TIM which is modelled from data presented in Paper III. Paper V deals with using deuterated sodium pyruvate as an alternative carbon source for protein perdeuteration. E. coli can utilize pyruvate as a carbon source and pyruvate-d3 can be made by relatively simple procedures. To circumvent the poor growth in minimal media with pyruvate as sole carbon source adaptive laboratory evolution was applied. Strains with improved growth in minimal medium with pyruvate-d3 as carbon source were obtained and shown to useful for production of perdeuterated proteins.

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