Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction

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

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

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.

Original languageEnglish
Article number13
Journalnpj Microgravity
Volume8
Issue number1
DOIs
StatePublished - Dec 2022

Funding

We are especially grateful for the assistance of April Spinale, Ray Polniak, and Marc Giulianotti, ISS National Laboratory, who provided guidance and oversight in flight preparation, which made this research possible. We thank Nicholas DeBouver and Ebuka Ogbouji for early work on the development of the TCB device. We also thank Nicholas DeBouver and Kevin Hill for assistance with PCG-8 and PCG-15 flight preparations, respectively. We also thank Lisa Keefe and Kevin Battaile for access and assistance at APS IMCA-CAT 17-ID-B. V.T.F. acknowledges the UK Engineering and Physical Sciences Research Council for grants EP/C015452/1 and GR/R99393/01 under which the Deuteration Laboratory was created within ILL’s Life Sciences Group. Funding was provided by the Center for the Advancement of Science in Space (contract GA2017-251, T.C.M. and C.A.S.) and the National Institutes of Health (1R01GM137008-01A1, T.C.M. and A.Y.K.). The research at ORNL’s High Flux Isotope Reactor (IMAGINE beamline) was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The authors thank the Institut Laue Langevin for provision of neutron beam time on the LADI-III beamline.

Fingerprint

Dive into the research topics of 'Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction'. Together they form a unique fingerprint.

Cite this