| 1. |
- Kelpšas, Vinardas, et al.
(författare)
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Enhancing protein perdeuteration by experimental evolution of Escherichia coli K-12 for rapid growth in deuterium-based media
- 2021
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 30:12, s. 2457-2473
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Tidskriftsartikel (refereegranskat)abstract
- Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild-type laboratory strain of E. coli K-12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi-generation adaptive laboratory evolution-based approach, we have isolated strains that show increased fitness in deuterium-based growth media. Whole-genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium-based media. By screening a collection of single-gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.
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| 2. |
- Kelpšas, Vinardas, et al.
(författare)
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Evolving escherichia coli host strains for efficient deuterium labeling of recombinant proteins using sodium pyruvate-d3
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 22:18
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Tidskriftsartikel (refereegranskat)abstract
- Labeling of proteins with deuterium (2H) is often necessary for structural biology tech-niques, such as neutron crystallography, NMR spectroscopy, and small-angle neutron scattering. Perdeuteration in which all protium (1H) atoms are replaced by deuterium is a costly process. Typ-ically, expression hosts are grown in a defined medium with heavy water as the solvent, which is supplemented with a deuterated carbon source. Escherichia coli, which is the most widely used host for recombinant protein production, can utilize several compounds as a carbon source. Glycerol-d8 is often used as a carbon source for deuterium labelling due to its lower cost compered to glucose-d7 . In order to expand available options for recombinant protein deuteration, we investigated the possibility of producing a deuterated carbon source in-house. E. coli can utilize pyruvate as a carbon source and pyruvate-d3 can be made by a relatively simple procedure. To circumvent the very poor growth of E. coli in minimal media with pyruvate as sole carbon source, adaptive laboratory evolution for strain improvement was applied. E. coli strains with enhanced growth in minimal pyruvate medium was subjected to whole genome sequencing and the genetic changes were revealed. One of the evolved strains was adapted for the widely used T7 RNA polymerase overexpression systems. Using the improved strain E. coli DAP1(DE3) and in-house produced deuterated carbon source (pyru-vic acid-d4 and sodium pyruvate-d3 ), we produce deuterated (>90%) triose-phosphate isomerase, at quantities sufficient enough for large volume crystal production and subsequent analysis by neutron crystallography.
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| 3. |
- Kelpšas, Vinardas, et al.
(författare)
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Neutron structures of Leishmania mexicana triosephosphate isomerase in complex with reaction-intermediate mimics shed light on the proton-shuttling steps
- 2021
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Ingår i: IUCrJ. - 2052-2525. ; 8:Pt 4, s. 633-643
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Tidskriftsartikel (refereegranskat)abstract
- Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. This simple reaction involves the shuttling of protons mediated by protolysable side chains. The catalytic power of TIM is thought to stem from its ability to facilitate the deprotonation of a carbon next to a carbonyl group to generate an enediolate intermediate. The enediolate intermediate is believed to be mimicked by the inhibitor 2-phosphoglycolate (PGA) and the subsequent enediol intermediate by phosphoglycolohydroxamate (PGH). Here, neutron structures of Leishmania mexicana TIM have been determined with both inhibitors, and joint neutron/X-ray refinement followed by quantum refinement has been performed. The structures show that in the PGA complex the postulated general base Glu167 is protonated, while in the PGH complex it remains deprotonated. The deuteron is clearly localized on Glu167 in the PGA-TIM structure, suggesting an asymmetric hydrogen bond instead of a low-barrier hydrogen bond. The full picture of the active-site protonation states allowed an investigation of the reaction mechanism using density-functional theory calculations.
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| 4. |
- Kelpšas, Vinardas, et al.
(författare)
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Perdeuteration, large crystal growth and neutron data collection of Leishmania mexicana triose-phosphate isomerase E65Q variant
- 2019
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Ingår i: Acta crystallographica. Section F, Structural biology communications. - 2053-230X. ; 75:4, s. 260-269
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Tidskriftsartikel (refereegranskat)abstract
- Triose-phosphate isomerase (TIM) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Two catalytic mechanisms have been proposed based on two reaction-intermediate analogues, 2-phosphoglycolate (2PG) and phosphoglycolohydroxamate (PGH), that have been used as mimics of the cis-enediol(ate) intermediate in several studies of TIM. The protonation states that are critical for the mechanistic interpretation of these structures are generally not visible in the X-ray structures. To resolve these questions, it is necessary to determine the hydrogen positions using neutron crystallography. Neutron crystallography requires large crystals and benefits from replacing all hydrogens with deuterium. Leishmania mexicana triose-phosphate isomerase was therefore perdeuterated and large crystals with 2PG and PGH were produced. Neutron diffraction data collected from two crystals with different volumes highlighted the importance of crystal volume, as smaller crystals required longer exposures and resulted in overall worse statistics.
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| 5. |
- Kelpsas, Vinardas
(författare)
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Production of Perdeuterated Proteins in Escherichia coli for Structural Studies by Neutron Crystallography
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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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| 6. |
- Kelpšas, Vinardas, et al.
(författare)
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Strain improvement of Escherichia coli K-12 for recombinant production of deuterated proteins
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Deuterium isotope labelling is important for structural biology methods such as neutron protein crystallography, nuclear magnetic resonance and small angle neutron scattering studies of proteins. Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affect growth of cells. The generation time for Escherichia coli K-12 in deuterated medium is substantially increased compared to cells grown in hydrogenated (protiated) medium. By using a mutagenesis plasmid based approach we have isolated an E. coli strain derived from E. coli K-12 substrain MG1655 that show increased fitness in deuterium based growth media, without general adaptation to media components. By whole-genome sequencing we identified the genomic changes in the obtained strain and show that it can be used for recombinant production of perdeuterated proteins in amounts typically needed for structural biology studies.
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