Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction

Journal Article · 04 May 2022 · npj Microgravity

DOI: https://doi.org/10.1038/s41526-022-00199-3 · OSTI ID:1866291

Drago, Victoria N. ; Devos, Juliette M. ; Blakeley, Matthew P. ; Forsyth, V. Trevor; Kovalevsky, Andrey Y. ; Schall, Constance A.; Mueser, Timothy C.

Abstract Biologically active vitamin B ₆ -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 ³ ). 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.

• Research Organization: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
• Sponsoring Organization: Center for the Advancement of Science in Space; National Institutes of Health (NIH); UK Engineering and Physical Sciences Research Council; USDOE; USDOE Office of Science (SC), Basic Energy Sciences (BES)
• Grant/Contract Number: AC05-00OR22725
• OSTI ID: 1866291
• Journal Information: npj Microgravity, Journal Name: npj Microgravity Journal Issue: 1 Vol. 8; ISSN 2373-8065
• Publisher: Nature Publishing GroupCopyright Statement
• Country of Publication: United States
• Language: English