Protein crystals were grown in a temperature controlled environment. This investigation's primary objective was to grow high quality, large crystals for ground-based research which were used in X-ray diffraction studies to discern the function and structure of the proteins.Principal Investigator(s)
Marshall Space Flight Center, Huntsville, AL, United States
National Aeronautics and Space Administration (NASA)Sponsoring Organization
Human Exploration and Operations Mission Directorate (HEOMD)Research Benefits
Information PendingISS Expedition Duration:
December 2001 - December 2002Expeditions Assigned
4,5Previous ISS Missions
Protein crystal growth investigations have been completed on STS-63, STS-67, STS-73, STS-83, STS-85, STS-94 and STS-95. PCG-STES investigations were conducted on ISS Increments 2 and 4 -11.
During Expedition 4, this investigation focused on the crystallization of human Replication Protein A (RPA) and manganese superoxide dismutase (SOD) to be used in ground-based X-ray diffraction studies to understand the atomic structure. Human RPA is a single-stranded DNA binding protein that is used in DNA metabolism (replication, transcription, recombination, and repair). SODs are antioxidant enzymes that protect living cells against oxide radicals that are associated with cell damage.
Samples were housed in the Protein Crystal Growth - Single Locker Thermal Enclosure System using the Protein Crystallization Apparatus for Microgravity (PCAM). The PCAMs consisted of nine trays, each containing seven vapor-equilibration wells. The nine trays were sealed inside a cylinder. Crystals were formed by the "sitting drop" method of vapor diffusion. Each sample well holds a drop of protein solution and precipitant (salts or organic solvents that draw water away from the protein solution) mixed together. A surrounding moat holds a reservoir, filled with an absorbent fluid, which draws moisture away from the mixed solution. Crystals formed as the moisture was absorbed. A rubber seal pressed into the lip of the reservoir kept the crystals from forming on Earth or from bouncing out of their wells during transport. This experiment had a total of 168 samples onboard ISS during two Expeditions inside the Single-locker Thermal Enclosure System (STES), making this an ideal method for mass crystal production.
The Single-locker Thermal Enclosure System (STES) provided a controlled-temperature environment between 1 degrees C and 40 degrees C to grow large, high-quality crystals. The thermal control system (TCS) regulated the temperature inside the payload chamber. Cabin air was pulled through an intake fan located on the front panel causing the air to flow across the heat exchanger fans, and then out the rear left side of the unit. Pushbuttons and an LCD display on the front panel allowed the crew to command the unit.
The crystals grown in microgravity are able to grow larger and more organized than those grown on Earth. The results from this investigation may further human space exploration efforts by creating technological and biological advancements as a direct result from this research.Earth Applications
Biotechnology and pharmaceutical researchers carry out the process of protein crystallization in order to grow large, well-ordered crystals for use in X-ray diffraction studies. However, on Earth, the protein crystallization process is hindered by forces of sedimentation and convection since the molecules in the crystal solution are not of uniform size and weight. This leads to many crystals of irregular shape and small size that are unusable for X-ray diffraction. X-ray diffraction is a complex process which requires several months to several years to complete, and the quality of data obtained about the three-dimensional structure of a protein is directly dependent on the degree of perfection of the crystals. Thus, the structures of many important proteins remain a mystery simply because researchers are unable to obtain crystals of high quality or large size. Consequently, the growth of high quality macromolecular crystals for diffraction analysis has been of primary importance for protein engineers, biochemists, and pharmacologists.
Fortunately, the microgravity environment aboard the ISS is relatively free from the effects of sedimentation and convection and provides an exceptional environment for crystal growth. Crystals grown in microgravity could help scientists gain detailed knowledge of the atomic, three-dimensional structure of many important protein molecules used in pharmaceutical research for cancer treatments, stroke prevention and other diseases. The knowledge gained could be instrumental in the design and testing of new drugs.
Crewmembers were required to transfer the PCG-STES from the Space Shuttle Middeck to ISS in EXPRESS Rack 4. Expeditions 4 used 4 STES units containing six PCAM cylinders in each unit. Each cylinder contained nine trays with seven reservoirs in each tray. PCG-STES-MM had 35 samples that were launched on December 5, 2001 and returned on April 19, 2002. Expedition 5 used 1 STES containing six PCAM cylinders. PCG-STES-MM had 133 samples for two proteins that were launched on October 18, 2002 and returned on December 7, 2002.
The experiment was activated by rotating the shaft end of the PCAM cylinder clockwise using a socket wrench. This causes the elastomer seal to retract allowing vapor diffusion between the protein solution and the crystallization solution, starting the experiment. For the deactivation, the cylinder is rotated counter clockwise to reseal the samples.
Crewmembers transferred the experiment hardware, PCG-STES (containing the PCAM chambers with the experiment samples) from the Space Shuttle Middeck to the ISS EXPRESS Rack 4. The experiment was activated by the crewmembers by opening the door on the STES unit and rotating the cylinder clockwise using a socket wrench. The crew checked the LCD display daily and cleaned the fan inlet when necessary. The samples were deactivated by rotating the cylinder counterclockwise using a socket wrench. For the return flight, the PCG-STES hardware was returned to the Shuttle for transport to Earth.
PCG-STES is a suite of nine experiments with additional shared samples for associated investigators. Samples were taken to and from station five times for crystallization during Expeditions 2, 4, 5, and 6. The logistical considerations of space flight affected some of the results, as flight delays compromised some samples, and a jarring drop of the hardware shortly after return on 11A/STS-113 probably destroyed any larger crystals that had formed during that set of runs. PCG-STES samples in DCAM were on orbit prior to the space shuttle Columbia accident, and then spent an unprecedented 981 days (Nov 2002 - Aug 2005) on ISS before being returned on the next space shuttle flight.
Not surprisingly, given the wide array of materials and objectives, some samples did produce large crystals, while other samples produced crystals no better than those produced on Earth. Yet other samples failed to crystallize at all.
Crystals of MnSOD, produced during Expedition 4, exhibited an 80-fold volume increase when compared to the crystals produced on Earth. The crystals that were produced in orbit ranged from small, needle-like crystals to large three-dimensional crystals. These crystals were used for Synchrotron X-ray analysis, the use of a high-energy, adjustable particle beam used for crystal diffraction. Through this analysis it was determined that the diffraction resolution and quality of data for the crystals produced in microgravity were increased when compared to the diffraction resolution of the crystals grown on Earth (Vahedi-Faridi et al. 2003).
High-resolution structural data were also obtained from human albumin and human antithrombin III crystals, and publications of new structural information is anticipated. Analyses of the samples returned in August 2005 is ongoing. (Evans et al. 2009)
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