Protein Crystal Growth-Single Locker Thermal Enclosure System-Crystallization of the Integral Membrane Protein Using Microgravity (PCG-STES-IMP) - 05.13.15
Protein crystals were grown in a temperature controlled environment. This investigation's primary objective was to grow high quality crystals for ground-based research which were to be used in understanding the structure of transporter proteins within cells. Science Results for Everyone
The objective of this investigation is to grow E. coli membrane protein crystals in microgravity, but none were produced. Analysis showed that protein drops added to sample trays were no longer present. These drops contained a detergent with a lower surface tension, which made them more sensitive to displacement than proteins without this detergent. The drops may have been displaced by a bump or jolt to the trays or the entire hardware unit at some point. Investigators concluded that new detergent recipes are needed in order to raise surface tension of the protein drops enough to survive normal movements associated with the trip to orbit and back. Experiment Details
Geoffrey Chang, Ph.D., University of California, La Jolla, CA, United States
NASA Marshall Space Flight Center, Huntsville, AL, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
Human Exploration and Operations Mission Directorate (HEOMD)
ISS Expedition Duration
June 2002 - December 2002
Previous 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.
- PCG-STES is comprised of nine separate investigations. They are: Improved Diffraction Quality of Crystals (IDQC), Integral Membrane Proteins (IMP), Measurements and Modeling (MM), Mitochondrial Metabolite Transport Proteins (MMTP), Material Science (MS), Ribosome for Diffraction Properties (RDP), Regulation of Gene Expression (RGE), Science and Applications (SA), and Vapor Equilibrium Kinetics Studies (VEKS).
- The objective of PCG-STES-IMP was to grow high-quality protein crystals for ground-based X-ray diffraction studies of the structure of bacteria transport proteins.
PCG-STES-RGE (Protein Crystal Growth - Single Locker Thermal Enclosure System - Regulation of Gene Expression) was performed during Expedition 5 and focused on the crystallization of two transporter proteins, Escherichia coli (E. coli) MsbA and EmrE. E. coli MsbA is a protein transporter responsible for transporting lipopolysaccharides and phospholipids from the inner membrane of the bacteria cell to the outer membrane, increasing the cell wall strength. It is theorized that antibiotics are transported out of the bacteria cells using this type of transporter protein and that knowledge of the structure of this protein could help develop a new class of drugs to supplement antibiotics. E. coli EmrE is of interest because its production is associated with antibiotic resistance.
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 is 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. Each cylinder holds 63 experiments for a total of 378 experiments 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.
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. Six PCAM cylinders were used. Each cylinder contained nine trays with seven reservoirs in each tray. PCG-STES-IMP had 63 samples.
The experiment is 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.
The PCG-STES-IMP operated on ISS during Expedition 5. The E. coli MsbA and EmrE membrane protein samples used did not produce crystals. Previous ground tests indicated that crystal growth was possible. Upon examining the pedestals (part of the PCAM trays where the sample was originally loaded) it was found that the protein drops were no longer present. The drops also contained a detergent that is used in crystallization experiments. These drops had a lower surface tension and were more sensitive to displacement than proteins that do not contain the detergent. An explanation for the displacement of the drops is that the trays, PCAMs, or the entire STES unit was bumped or jolted at some point before the experiment was initiated.
The investigator has concluded that new recipes for crystallization that use detergents are needed in order to raise the surface tension of the protein drops so they can survive the normal movements associated with STES unit during the round trip from ground to orbit. (Chang, One Year PostflightReport, 2003)
Ground Based Results Publications
Declercq J, Evrard C, Carter DC, Wright BS, Etienne G, Parello J. A crystal of a typical EF-hand protein grown under microgravity diffracts X-rays beyond 0.9 Å resolution. Journal of Crystal Growth. 1999; 196(2-4): 595-601. DOI: 10.1016/S0022-0248(98)00829-X.
Ho JX, Declercq J, Myles DA, Wright BS, Ruble JR, Carter DC. Neutron structure of monoclinic lysozyme crystals produced in microgravity. Journal of Crystal Growth. 2001; 232(1-4): 317-325. DOI: 10.1016/S0022-0248(01)01077-6.
Carter DC, Wright BS, Miller T, Chapman J, Twigg P, Keeling K, Moody K, White M, Click J, Ruble JR, Ho JX, Adcock-Downey L, Dowling T, Chang C, Ala P, Rose J, Wang BC, Declercq J, Evrard C, Rosenberg J, Wery J, Clawson D, Wardell M, Stallings W, Stevens A. PCAM: a multi-user facility-based protein crystallization apparatus for microgravity. Journal of Crystal Growth. 1999; 196: 610-622. DOI: 10.1016/S0022-0248(98)00858-6.
Zörb C, Weisert A, Stapelmann J, Smolik G, Carter DC, Wright BS, Brunner-Joos KD, Wagner G. Bacteriorhodopsin crystal growth in reduced gravity--results under the conditions, given in CPCF on board of a Space Shuttle, versus the conditions, given in DCAM on board of the Space Station Mir. Microgravity Science and Technology. 2002; 13(3): 22-29. DOI: 10.1007/BF02872073. PMID: 12206160.
NASA Fact Sheet
NASA Image: ISS007E14210 - Close-up of the Single-locker Thermal Enclosure System in Express Rack 4 onboard ISS, during Expedition 7.
+ View Larger Image
NASA Image: ISS005E21531 - Astronaut Peggy A. Whitson, Expedition Five science officer, works the PCG-STES hardware on board the International Space Station.
+ View Larger Image