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Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction

Drago, Victoria N. ; Devos, Juliette M. ; Blakeley, Matthew P. ; Forsyth, V. Trevor LU ; Kovalevsky, Andrey Y. ; Schall, Constance A. and Mueser, Timothy C. (2022) In npj Microgravity 8(1).
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... (More)

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.

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author
; ; ; ; ; and
organization
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type
Contribution to journal
publication status
published
subject
in
npj Microgravity
volume
8
issue
1
article number
13
publisher
Nature Publishing Group
external identifiers
  • scopus:85129650117
  • pmid:35508463
ISSN
2373-8065
DOI
10.1038/s41526-022-00199-3
language
English
LU publication?
yes
id
2e450b0b-7ac5-46c2-add0-89a50c0b160d
date added to LUP
2022-07-05 14:40:30
date last changed
2024-04-02 13:20:14
@article{2e450b0b-7ac5-46c2-add0-89a50c0b160d,
  abstract     = {{<p>Biologically active vitamin B<sub>6</sub>-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 mm<sup>3</sup>). 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.</p>}},
  author       = {{Drago, Victoria N. and Devos, Juliette M. and Blakeley, Matthew P. and Forsyth, V. Trevor and Kovalevsky, Andrey Y. and Schall, Constance A. and Mueser, Timothy C.}},
  issn         = {{2373-8065}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Nature Publishing Group}},
  series       = {{npj Microgravity}},
  title        = {{Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction}},
  url          = {{http://dx.doi.org/10.1038/s41526-022-00199-3}},
  doi          = {{10.1038/s41526-022-00199-3}},
  volume       = {{8}},
  year         = {{2022}},
}