The Effect of Microgravity on the Co-crystallization of a Membrane protein with a medically relevant compound. (CASIS PCG 4-2) - 07.14.16

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Scientists crystallize proteins to understand how these large, complex molecules function and how they are arranged, which can help them design better drugs. The Effect of Microgravity on the Co-crystallization of a Membrane protein with a medically relevant compound (CASIS PCG 4-2) investigation crystallizes a human membrane protein in the presence of a medically relevant compound. Microgravity enables the formation of larger, more perfect crystals than those grown on Earth, enabling a three-dimensional view of the protein’s structure that allows scientists to design special drugs to target a biological pathway involved in cancer.
Science Results for Everyone
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The following content was provided by Michael John Hickey, and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom:

Principal Investigator(s)
Michael John Hickey, Eli Lilly and Company, San Diego, CA, United States

Co-Investigator(s)/Collaborator(s)
Margaret Kearins, Eli Lilly and Company, San Diego, CA, United States
Xun Zhao, Eli Lilly and Company, San Diego, CA, United States
Joseph Ho, Eli Lilly and Company, San Diego, CA, United States

Developer(s)
Center for the Advancement of Science in Space (CASIS), Rockledge, FL, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Earth Benefits, Scientific Discovery

ISS Expedition Duration
March 2016 - September 2016

Expeditions Assigned
47/48

Previous Missions
Information Pending

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Experiment Description

Research Overview

It is believed that the diffraction quality of membrane protein co-crystals grown on earth can be adversely affected, among other things, by convective mixing and sedimentation during the equilibration process. As a result, the growing crystal lattice can accumulate defects and irregularities that can negatively affect crystal diffraction –as can be evidenced by a net higher mosaicity and lower resolution limit.
 
Co-crystallization of membrane proteins in microgravity (i.e., on the International Space Station) may allow for a slower rate of addition of macromolecules to the growing and extending crystal lattice(s). This, in theory, would allow more time for crystal lattice alignment, the minimization of forming lattice defects and irregularities that can be caused by incorporating impurities. Improved co-crystal quality, uniformity, packing, etc. in microgravity would be evidenced by higher resolution limits and lower mosaicity values, as compared to similar earth-based experimentation.
 
A human membrane protein is to be co-crystallized with a medically relevant compound in identical experiments both in microgravity and 1g:
  1. Optimization experiments: A. 30 vapor diffusion experiments, in an HDPCG device, are set up at ambient temperature around the single best co-crystallization condition(s) for the membrane proteins, and compounds, that has yielded crystals at 1g.
  2. Broad Screening experiments: A. 192 vapor diffusion experiments, in a 96 well plate-format, are set up in duplicate at ambient temperature (after freeze-thaw) around several co-crystallization conditions that have yielded crystals at 1g.
Crystals grown both in microgravity and in earth-based controls/experiments are evaluated both qualitatively (size, shape, number, etc.) and quantitatively (diffraction, resolution, mosaicity, etc.).
 
It is anticipated that improved crystal sizes, packing, and more importantly higher resolution and reduced mosaicity of microgravity grown co-crystals (membrane protein with compound) over earth-grown co-crystals. This helps to better understand and define the binding site of the medically relevant compound to the macromolecule. In addition, a better resolved 3D structure helps facilitate protein construct design, which could help improve earth based co-crystallization experiments and diffractive quality of crystals. Overall, the structure(s) enables and facilitates structure-based drug design (SBDD) on a cancer target that has yet to be exploited medicinally.

Description

Since the 1990’s, structure-based drug design (SBDD) has been an integral component in the drug discovery and development process. One of the greatest obstacles that still needs to be overcome and to facilitate this process has been the inability to co-crystallize various macromolecular proteins with and without specific ligands (i.e., co-factors, nucleic acids, etc). Protein crystallization has traditionally been considered challenging for a number of reasons - including the restrictions of the aqueous environment, difficulties in obtaining high-quality protein samples, protein concentration limits, temperature, pH, ionic strength, etc. In addition, the effects of sedimentation and convective mixing that can adversely affect growing crystals in vapor diffusion experiments can often present a formidable challenge in obtaining high-resolution diffraction and structures. Given that proteins have their own unique physicochemical characteristics, the crystallization of a particular protein is rarely predictable utilizing Earth-based crystallization methods.
 
The crystallization of membrane proteins creates an even more challenging effort since, in addition to hydrophilic surfaces, these macromolecules have hydrophobic surfaces (thereby necessitating detergents, additives, etc), they are relatively flexible, and are typically expressed at relatively low levels. This creates difficulties in obtaining enough protein via purification methods suitable enough to grow crystals, and can make determining atomic resolution structures for these macromolecules more difficult than for globular proteins.
 
The membrane proteins selected to co-crystallize with a medically relevant compound for which exists low resolution diffraction data. The final co-crystal sizes, quality and nucleation sites within the drops leads us to believe that sedimentation and convective mixing are adversely affecting membrane protein crystal growth.
 
It is believed that (co)crystallization in microgravity would provide an extremely useful means of overcoming the sedimentation and convection obstacles that can be typically encountered in Earth-based vapor diffusion experiments. The Handheld High Density Protein Crystal Growth (HDPCG) apparatus, and broad screening with MiTiGen plates, provide a platform to explore this theory.

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Applications

Space Applications
In space, convection and gravity do not interfere with crystal formation, yielding larger and higher-quality protein crystals. Scientists can use X-ray techniques to study large crystals and determine how protein molecules are organized. This investigation crystallizes a human membrane protein that has been shown to be involved in several types of cancer. The investigation also demonstrates the ongoing commitment of NASA to facilitate technological advances on the International Space Station. Even if the investigation is unsuccessful, the outcome will provide lessons for improved protein crystallization techniques, benefiting future space-based research.

Earth Applications
With a greater understanding of protein structures, scientists can design drugs with specific molecular shapes so they interact with proteins in specific ways. This investigation crystallizes a human membrane protein, one of many large molecules inside human cells that are important targets for pharmaceuticals. The investigation focuses on a known cancer target, so improving structure-based drug design could benefit many patients suffering from cancer.

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Operations

Operational Requirements and Protocols

  • Time between turnover and launch: L-36 hours
  • HDPCG hardware:
  • Ascent: 
    • Launches at ambient 21°C, +/- 2°C
  • On-orbit:
    • Stowage at 21°C, +/- 2°C
    • Sample activation to be completed on ISS
    • Sample deactivation can occur within 30 days following activation
  • Descent:
    • Temperature at 21°C, +/- 2°C
    • Early recovery at the dock desired, samples are picked up by PI.
  • MiTiGen Plates:
    • Ascent:
      • Launches frozen at -80°C
    • On-orbit:
      • Stowage at 21°C once aboard ISS.
    • Descent:
      • Temperature at 21°C, +/- 2°C
    • Early recovery at the dock desired, samples are picked up by Principal Investigator.

  1. The Handheld HDPCG assembly is transferred from the ascent vehicle at 21°C to ISS. To activate the experiment samples in the Handheld HDPCG hardware, an Activation Tool (which is attached to the side of the HDPCG hardware using Velcro) is removed and attached to each of the five cell blocks. Each cell block is turned 90 degrees clockwise to align the sample insert with the precipitant solution. Crystals grow for 21 – 30 days. The Handheld HDPCG assembly is deactivated. Deactivation is accomplished by attaching the Activation Tool to each cell block and turning 180° clockwise to turn the protein insert opposite of the precipitant reservoir. The Handheld HDPCG assembly is ready then for descent.
  2. The MiTiGen plates need to be transferred from the ascent vehicle at -80°C to ISS. They only need to be transferred to ambient 21°C to become activated. Crystals grow for 21 – 30 days, then plates are ready for descent at 21°C.

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Decadal Survey Recommendations

Information Pending

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Results/More Information

Information Pending

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Related Websites
Eli Lilly and Company

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Imagery