Commercial Protein Crystal Growth - High density protein crystal growth Modified (CPCG-HM) - 03.25.14

Overview | Description | Applications | Operations | Results | Publications | Imagery
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The Commercial Protein Crystal Growth HM (CPCG-HM) investigation expands an ongoing program into the complex realm of membrane proteins that move signals or molecules to and from a cell’s interior or help cells identify each other for immune responses. Proteins in the microgravity environment are exposed to conditions that concentrate them so they form crystals that would be too fragile to form on Earth, but which can be returned to Earth for X-ray analysis. 

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Information Pending



This content was provided by Lawrence J. DeLucas, Ph.D., O.D., and is maintained in a database by the ISS Program Science Office.

Experiment Details

OpNom

Principal Investigator(s)

  • Lawrence J. DeLucas, Ph.D., O.D., University of Alabama at Birmingham, Birmingham, AL, United States

  • Co-Investigator(s)/Collaborator(s)
  • Joseph D. Ng, Ph.D., iXpressGenes, Hunstville, AL, United States
  • Alexander McPherson, Ph.D., University of California, Irvine, Irvine, CA, United States

  • Developer(s)
    University of Alabama, Birmingham, Birmingham, AL, 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 2014 - September 2014

    Expeditions Assigned
    39/40

    Previous ISS Missions
    • HDPCG trays flew installed in a Commercial Refrigerator Incubator Module (CRIM) during Expeditions 2 and 4.
    • PCG Straws flew installed in a Sample Transfer Container (STC) during Russian Soyuz flights TMA-13 and TMA-16.
    • MERLIN hardware that will support the CPCG-HM experiment has operated continuously on ISS since Nov 2008. 
     

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    Experiment Description

    Research Overview

    • To conclusively demonstrate the effect of microgravity on the size and quality of crystals for a variety of high-value proteins.


    • Upon completion for the experiments it will be demonstrated that microgravity protein crystallization provided an improvement over ground controlled crystallization.


    • Improved space grown protein crystals will provide three-dimensional x-ray crystallographic structures for several high valued proteins. Eventually these structures will help researchers determine how specific proteins function and are involved in the disease process. Numerous drugs today are the product of this structured based drug design.

    Description

    Proteins are important macromolecules without which our bodies would be unable to repair, regulate, or protect themselves. The use of X-ray crystallography to determine protein structure requires the production of well-ordered protein crystals that are of sufficient quality. Without high quality crystals of a protein, it is impossible to carry out crystallographic structural studies. Using three-dimensional structure information, researchers can determine how proteins function and in cases where these proteins are involved in disease processes, the structure is often used to design new drugs that specifically interact with the protein. Many leading drugs today are the product of structure-based drug design. 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.

    While enormous strides have been made in the last decade, there remain a large number of important proteins where the difficulty of obtaining high-quality crystals is the chief barrier to their structural analysis. One class of proteins, membrane proteins, comprises a number of targets identified by the pharmaceutical industry as high-value commercial opportunities (membrane proteins were the targets for approximately 67% of all past marketed drugs and it is estimated that they will comprise targets for an equal percentage of all future drugs)[1]. Membrane protein crystallization represents one area where the onset of space commercialization can maximize the impact of the microgravity environment in space-based research applications for the academic, government and pharmaceutical industry. Other areas include high-value aqueous proteins and protein complexes (based on their functional importance in biological systems) many of which have yielded crystals of poor quality (thus far microgravity crystallization has been attempted on only 3 membrane proteins and two protein complexes). Access to unique data optimized in microgravity could have great relevance for understanding protein structures and advancing new drugs into the pharmaceutical market.
    Crystal growth in a microgravity environment can have beneficial effects on the size and more importantly, the intrinsic order of these protein crystals. Membrane proteins and large aqueous proteins or protein complexes typically diffract poorly and exhibit high mosaicity.

    Significant disadvantages of past microgravity flights include the short mission duration (the majority of the past data was collected on spatial flights with mission durations of two weeks or less). The results of the program, while intriguing, had a limited impact on structural biology during a time when technological innovations on the ground have produced significant and fundamental advances in our understanding of protein function. However, despite the increased sophistication of ground-based protein crystallization projects, the crystals of a large number of important targets today still have suboptimal diffraction characteristics. Even a slight improvement in diffraction data would have a significant impact on scientist’s ability to use the resulting structures to provide important insights into biological mechanisms.

     

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    Applications

    Space Applications

    While Earth-based protein crystal studies have made significant advances in recent years; details of large, key membrane proteins remain hidden in X-ray studies. Even with modest improvements from crystals grown in space without the defects caused by gravity, should allow significant advances in understanding their structure and how they function. 

    Earth Applications

    High-density membrane proteins are targeted for about two-thirds of the current and anticipated pharmaceutical market. Pure crystals that yield information about the protein’s structure will open the way for a coherent, structure-based design of a broader range of medicines for treating diseases and disorders.  

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    Operations

    Operational Requirements

    • Pre-flight late loading (~L-24 hours) and post-flight early access (R+48) required

    • 28V power required during all mission phases (pre-flight, ascent, ISS, descent, post-flight) to maintain set-point temperatures and prevent loss of science.

    • Minimal crew time: ~ 180 minutes

      • 60 minutes to transfer two CPCG-HM units from ETOV to EXPRESS Rack

      • 30 minutes at beginning of mission to activate HDPCG trays (4)

      • 30 minutes at end of mission to deactivate HDPCG trays (4)

      • 60 minutes to transfer two CPCG-HM units from EXPRESS Rack to Dragon

      • PCG Straws are frozen pre-flight and self-activate upon thaw (no crew time required)

      • Each CPCG-HM replaces one ISS locker, weighing

    • Each CPCG-HM will interface with the AAA loop.
       

    Operational Protocols

     

    NOMINAL ACTIVITIES

    • TRANSFER FROM ETOV to EXPRESS AND SETUP
    • ACTIVATION

    -  Slide Mechanism to Hard Stop Left or Hard Stop Right (Launch Location Dependant) (8)

        will align Protein Insert with PPT Reservoir

    • DEACTIVATION

    -  Slide Mechanism to Hard Stop Left or Hard Stop Right (Opposite Activation Direction) (8)

        will align Protein Insert with Access Cap

    • STATUS CHECK (Laptop or Front Panel)
      •  Twice daily (only if no KU for 24 hours; 8 to 12 hour desired separation)
      •  Check temperature is within ± 2°C of set point temperature
      •  Check for error messages **
    • PHOTO (Crew activating and deactivating CPCG-HM)
    • TRANSFER FROM EXPRESS TO DRAGON

    ALTERNATE ACTIVITIES (MERLIN)

    • RECONFIGURE
    • POWER UP
    • WARM SHUTDOWN
    • REBOOT
    • MERLIN Laptop Application Installation (if required for additional MERLIN)
    • MERLIN Setup (if move to alternate rack or another unit arrives)

    MALFUNCTION OPERATIONS (MERLIN)

    • ERROR MESSAGE
    • RECORD DIAGNOSTIC DATA

     

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    Results/More Information

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    Related Websites
    uab.edu

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    Imagery

    image CPCG-HM Design ? Illustration depicts the MERLIN Assembly with 2 HDPCG trays and STC tray that will hold the liquid-to-liquid sample straws. Image courtesy of University of Alabama.
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    The comparison of protein crystal, Bovine Insulin space-grown (left) and earth-grown (right) from PI Larry DeLucas' previous microgravity research. Image courtesy of NASA.

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    image CPCG-HM Design ? Illustration the CPCG-HM Assembly with 4 HDPCG trays. Image courtesy of University of Alabama.
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    image CPCG-HM Design ? Vapor Diffusion Sample Blocks configuration from Launch to Landing. Image courtesy of University Alabama.
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    image CPCG-HM Design ? Crystal formation inside Vapor Diffusion Sample Block. Image courtesy of University of Alabama.
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