Comparative analysis of anti-polyglutamine Fab crystals grown on Earth and in microgravity

Creators: Owens, Gwen E.; New, Danielle M.; Olvera, Alejandra I.; Manzella, Julia Ashlyn; Macon, Brittney L.; Dunn, Joshua C.; Cooper, David A.; Rouleau, Robyn L.; Connor, Daniel S.; Bjorkman, Pamela J.

Abstract

Huntington's disease is one of nine neurodegenerative diseases caused by a polyglutamine (polyQ)-repeat expansion. An anti-polyQ antigen-binding fragment, MW1 Fab, was crystallized both on Earth and on the International Space Station, a microgravity environment where convection is limited. Once the crystals returned to Earth, the number, size and morphology of all crystals were recorded, and X-ray data were collected from representative crystals. The results generally agreed with previous microgravity crystallization studies. On average, microgravity-grown crystals were 20% larger than control crystals grown on Earth, and microgravity-grown crystals had a slightly improved mosaicity (decreased by 0.03°) and diffraction resolution (decreased by 0.2 Å) compared with control crystals grown on Earth. However, the highest resolution and lowest mosaicity crystals were formed on Earth, and the highest-quality crystal overall was formed on Earth after return from microgravity.

Additional Information

© 2016 International Union of Crystallography. Received 13 June 2016. Accepted 2 September 2016. Edited by R. L. Stanfield, The Scripps Research Institute, USA. We thank the Gordon and Betty Moore Foundation for support of the Molecular Observatory at Caltech. The operations at SSRL are supported by the Department of Energy and by the National Institutes of Health. GEO was supported by National Research Service Awards (T32GM7616 and T32GM08042) from the National Institute of General Medical Sciences and by the UCLA–Caltech Medical Scientist Training Program. GEO, DMN and AIO were supported by the Center for Advancement of Science in Space (CASIS PCG HDPCG-1). GEO and PJB conceived the study, GEO, AIO and DMN performed protein expression and purification, GEO collected X-ray diffraction data, GEO, DC, AM, BM, RR and JD set up experiments at KSC prior to the SpaceX-3 launch, GEO and DC retrieved samples after the return of SpaceX-4 to Earth, DMN analyzed crystal size data, and GEO and PJB wrote the paper, with all co-authors contributing to scientific planning and discussions. We thank Dr Robert Hughes for the GFP-huntingtin construct, Dr IhnSik Seong for the full-length huntingtin protein, Beth Huey-Tubman and Allen Lee for technical support and encouragement, Dr Jennifer Keeffe for scientific guidance, Marta Murphy for assistance with figures, the Protein Expression Center at Caltech for purification of 3B5H10 Fab, the scientific staff of SSRL beamline 12-2 for assistance with X-ray diffraction experiments and the members of the Bjorkman laboratory for critical reading of the manuscript. We thank Drs Abebe Hassen and Fred Owens for assistance with statistical analysis. We thank the team at CASIS for support, particularly April Spinale and Ken Shields. We would also like to thank Scott Slack and his film team at High Impact and Dr Edward Wild for helping to make our research accessible to a broad audience. We also thank the two astronauts, Steve Swanson and Reid Wiseman, who operated our experiments on the ISS.

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