Crystallization of Huntingtin Exon 1 Using Microgravity (CASIS PCG HDPCG-1) - 03.29.17
The Crystallization of Huntingtin Exon 1 Using Microgravity (CASIS PCG HDPCG-1) investigation focuses on the crystallization of huntingtin, a protein associated with Huntington’s disease. Crystallization is crucial for the development of new drugs to treat this degenerative brain disorder, which is caused by an inherited mutation in the huntingtin gene. But the huntingtin protein has evaded crystallization for more than a decade. Crystallizing it would be an important milestone in the field of macromolecular crystallography as well as a step toward developing a treatment for this ultimately fatal disease.
Science Results for Everyone
The hunt goes on. Huntington’s disease is one of nine neurodegenerative diseases related to an amino acid, polyglutamine (polyQ). Attempts to crystallize PolyQ and anti-polyQ antibodies called MW1 aboard the space station resulted in crystals of MW1, but analysis shows fewer suitable crystals grew in microgravity than on Earth. Results also confirm that crystal forms have little effect on the quality of crystals grown in microgravity. Crystallizing proteins in space remains a challenge and more experiments are needed to compare microgravity-grown crystals with the best ground-based crystals to determine the relative value of microgravity protein crystallization. Experiment Details
OpNom: Handheld HDCPG
Pamela Bjorkman, Ph.D., California Institute of Technology, Pasadena, CA, United States
University of Alabama at Birmingham, Birmingham, AL, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
National Laboratory (NL)
ISS Expedition Duration
March 2014 - September 2014
Huntington’s disease is a fatal neurodegenerative disorder with no known cure. Crystallization of huntingtin, which has evaded crystallization for more than a decade, would be an important milestone in the field of macromolecular crystallography. The crystallization of huntingtin is needed because it is crucial for development of new drugs for the treatment of Huntington’s disease and other related disorders.
Researchers plan to grow crystals of medically relevant proteins to accurately determine their shape to fit new drugs. The proteins chosen are proving extremely difficult to crystalize on Earth and it is believed that zero-gravity will allow the crystals to grow. This investigation crystallizes huntingtin exon 1 using the unique microgravity environment to increase the crystal quality and size compared to protein crystals grown on Earth.
Because huntingtin is part of a larger class of amyloidogenic proteins whose crystal structures have also never been solved, the crystallization of huntingtin would be a scientific breakthrough in crystallization of these difficult proteins. Commercial impact of crystallization of huntingtin includes potential development of pharmaceutical agents that specifically bind to the structure of mutant huntingtin.
In microgravity, the elimination of sedimentation and convection produces a highly unique environment for space-based experiments such as protein crystallization. The human body contains over 100,000 proteins that play important roles in the everyday function of the body such as the formation of major components of muscle and skin, and how the body fights diseases. In order to fully understand the function of proteins, three-dimensional structural information becomes necessary. The structure of individual proteins can be studied with the growth of high quality crystals in which the molecules of the protein are arranged in a regular, repeating pattern. In order to produce high-quality crystals of a protein, it must be reasonably pure with respect to other contaminating proteins, it must be homogeneous and it's three-dimensional conformation relatively stable. Crystallization occurs in aqueous solution when purified protein molecules are coaxed to slowly self-associate, through relatively weak interactions such as ionic or hydrogen bonds. Individual protein molecules align themselves in a repeating series of "unit cells" by adopting a consistent orientation that eventually forms a crystal with sharp facets. Protein crystallization serves as the basis for X-ray crystallography, wherein a crystallized protein is used to determine the protein’s three-dimensional structure via x-ray diffraction. This investigation aims to successfully crystallize the first complete structure of huntingtin exon 1 using microgravity, alone and in complex with binding proteins to stabilize the pathogenic polyglutamine regions of huntingtin. A structure of huntingtin would address a critical problem in engineering new therapeutics to treat or prevent Huntington’s disease- the absence of a complete X-ray crystal structure of the pathologic region of huntingtin. Crystallization is performed using the Vapor Diffusion technique inside the Handheld HDPCG. This technique is also known as the hanging drop method of crystal growth. This process entails a droplet containing purified protein, buffer, and precipitant being allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water vaporizes from the drop and transfers to the reservoir, the precipitant concentration increases to a level optimal for crystallization.
In general, crystals grown in microgravity are able to reach much larger sizes and more perfect forms than those grown on Earth, because the effects of gravity are removed. Crystallizing proteins allows scientists to determine how they are structured, and thereby how a new drug might interact with them. Existing processes have been unable to crystallize proteins that are prone to clumping together, including huntingtin and the amyloid-beta protein involved in Alzheimer’s disease. This investigation could yield a new area of space-based research in protein crystallography, especially for proteins whose structure or size makes them difficult or impossible to crystallize on Earth. In addition, if this investigation demonstrates that several important proteins only crystallize in microgravity, a new multi-billion-dollar market could emerge for development of highly effective and specific treatments.
Using a three-dimensional crystal, scientists may be able to identify a potential drug treatment for Huntington’s disease. Scientists aim to identify specific sections of the protein that might interact with other compounds. This could produce a new targeted drug that works efficiently with few side effects, improving the quality of life for patients with Huntington’s disease. Additionally, new insight into the physics of crystal formation in microgravity could lead to new approaches in crystallography on Earth, including for several other medically relevant proteins.
Operational Requirements and Protocols
- Preflight late loading (~L-24 hours) and postflight early access (R+48) required
- Temperature Controlled environment (+4 Deg C +/- 2 Deg C) for turnover, launch, on-orbit and return.
Minimal crew time: ~ 60 minutes
- 30 minutes for access from cold stowage and sample activation (2 blocks)
- 30 minutes at end of mission to deactivate (2 Blocks) and stow in Cold stowage for return.
- Quiescent microgravity environment during the sample activated period.
- Cold Stowage unpacking
- HH HDPCG Activation
- HH HDPCG Deactivation
Cold Stowage packing
Decadal Survey Recommendations
Information Pending^ back to top
Huntington's disease (HD) is an inherited disease in which nerve cells in the brain waste away resulting progressively in severe irreversible mental and physical disabilities. It is one of nine neurodegenerative diseases caused by a polyglutamine (polyQ)-repeat expansion. Like Lego bricks, amino acids are building blocks of protein in the body, and polyQ is a portion of a protein consisting of a sequence of several units of the amino acid Glutamine. PolyQ and anti-polyQ antibodies (MW1), proteins associated with HD, were grown both on Earth and on the International Space Station, where microgravity conditions could enhance the quality of the protein crystals for research. Huntington’s polyQ was not crystallized in microgravity, but crystals of MW1 were readily obtained. Once the crystals returned to Earth, the number, size, and appearance of all crystals were recorded, and X-ray crystal structural data were collected. 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 (high mosaicity, or lattice defects, can interfere with measurements of crystal structure) and diffraction resolution compared with control crystals grown on Earth. Microgravity-grown crystals showed improved X-ray diffraction resolution on average, but the highest-quality crystal overall was formed on Earth. Quantitative analyses of crystal number and visible crystal area from microscopy images demonstrated that fewer crystals of a size suitable for diffraction were grown per well in microgravity compared with ground controls. Results also reaffirm that crystal forms do not play a major role in whether or not crystals grow better in microgravity. While experiments show that ISS is a potential platform for crystal growth, crystallization of proteins in space remains a challenge. Given the expense and time involved in crystallization trials in microgravity, future experiments should consider the potentially deleterious effects of fluid convection on vapor-diffusion crystallization experiments. Additionally, a comparison of microgravity-grown crystals with the best crystals obtainable through ground-based methods is necessary to realistically determine the relative value of microgravity protein crystallization.^ back to top
Owens GE, New DM, Olvera AI, Manzella JA, Macon BL, Dunn JC, Cooper DA, Rouleau RL, Connor DS, Bjorkman PJ. Comparative analysis of anti-polyglutamine Fab crystals grown on Earth and in microgravity. Acta Crystallographica Section F: Structural Biology Communications. 2016 October 1; 72(10): 762-771. DOI: 10.1107/S2053230X16014011. PMID: 27710941.
Ground Based Results Publications
NASA Image: ISS041E078028 - NASA astronaut Reid Wiseman retrieving Ambient Handheld High Density Protein Crystal Growth (HDPCG) Unit
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NASA Image: ISS041E078032 - NASA astronaut Reid Wiseman retrieving Ambient Handheld High Density Protein Crystal Growth (HDPCG) Unit
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NASA Image: ISS041E078040 - NASA astronaut Reid Wiseman retrieving Ambient Handheld High Density Protein Crystal Growth (HDPCG) Unit.
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