Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction
Abstract Biologically active vitamin B6-derivative pyridoxal 5′-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. S...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-05-01
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| Series: | npj Microgravity |
| Online Access: | https://doi.org/10.1038/s41526-022-00199-3 |
| _version_ | 1827614437938298880 |
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| author | Victoria N. Drago Juliette M. Devos Matthew P. Blakeley V. Trevor Forsyth Andrey Y. Kovalevsky Constance A. Schall Timothy C. Mueser |
| author_facet | Victoria N. Drago Juliette M. Devos Matthew P. Blakeley V. Trevor Forsyth Andrey Y. Kovalevsky Constance A. Schall Timothy C. Mueser |
| author_sort | Victoria N. Drago |
| collection | DOAJ |
| description | Abstract Biologically active vitamin B6-derivative pyridoxal 5′-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm3). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15. |
| first_indexed | 2024-03-09T08:54:58Z |
| format | Article |
| id | doaj.art-33403e48882b426c846ca86ebca63f25 |
| institution | Directory Open Access Journal |
| issn | 2373-8065 |
| language | English |
| last_indexed | 2024-03-09T08:54:58Z |
| publishDate | 2022-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | npj Microgravity |
| spelling | doaj.art-33403e48882b426c846ca86ebca63f252023-12-02T13:14:06ZengNature Portfolionpj Microgravity2373-80652022-05-01811910.1038/s41526-022-00199-3Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffractionVictoria N. Drago0Juliette M. Devos1Matthew P. Blakeley2V. Trevor Forsyth3Andrey Y. Kovalevsky4Constance A. Schall5Timothy C. Mueser6Department of Chemistry and Biochemistry, University of ToledoLife Sciences Group, Institut Laue-LangevinLarge-Scale Structures Group, Institut Laue-LangevinLife Sciences Group, Institut Laue-LangevinNeutron Scattering Division, Oak Ridge National LaboratoryDepartment of Chemical Engineering, University of ToledoDepartment of Chemistry and Biochemistry, University of ToledoAbstract Biologically active vitamin B6-derivative pyridoxal 5′-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm3). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15.https://doi.org/10.1038/s41526-022-00199-3 |
| spellingShingle | Victoria N. Drago Juliette M. Devos Matthew P. Blakeley V. Trevor Forsyth Andrey Y. Kovalevsky Constance A. Schall Timothy C. Mueser Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction npj Microgravity |
| title | Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction |
| title_full | Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction |
| title_fullStr | Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction |
| title_full_unstemmed | Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction |
| title_short | Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction |
| title_sort | microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction |
| url | https://doi.org/10.1038/s41526-022-00199-3 |
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