Advanced Protein Crystallization Facility - Protein Crystallization in Microgravity, Collagen Model (X-Y-Gly) Polypeptides - the case of (Pro-Pro-Gly) 10 (APCF-PPG10) - 07.14.16
Specialized microgravity facility that offered researchers several different crystal growth options in a controlled environment that enabled undisturbed nucleation (beginning of chemical changes at discrete points in a system) and growth of proteins to obtain large crystals for analysis on Earth. Understanding the results obtained from the crystals will lead to advances in manufacturing and biological processes. Science Results for Everyone
This investigation grew Pro-Pro Gly10, or PPG10, a collagen protein crystal found in concentrated skin, joints, and bone tissue. As expected, these space-grown crystals were superior to those grown on Earth. This investigation allowed direct comparison of four different crystallization environments—solution and agarose in microgravity and on earth – and researchers determined for the first time a direct correlation between crystal motion and International Space Station acceleration events such as docking, venting, and crew movement. This motion affected crystal distribution but there appeared to be no correlation with quality, suggesting that crystal growth is actually controlled by motion. Experiment Details
Adriana Zagari, Ph.D., University of Naples, Naples, Italy
Astrium GmbH, Bremen, Germany
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
Italian Space Agency (ASI)
ISS Expedition Duration
August 2001 - December 2001
The APCF has flown on several Shuttle flights dating back to 1985 including, STS-107 (Columbia), which was lost in 2003.
- The APCF hardware contained 8 separate protein crystal investigations; these included APCF-Camelids, APCF-Crystal Quality, APCF-Crystal Growth, APCF-Lipoprotein, APCF-Lysozyme, APCF-Octarellins, APCF-PPG10 and APCF-Rhodopsin.
- PPG10 crystals were observed for growth and motion during the four month long ISS Expedition 3 mission. APCF-PPG10 allowed, for the first time, the direct comparison of four different crystallization environments: solution in microgravity, agarose gel in microgravity, solution on Earth and agarose gel on Earth as well as a comparison between two microgravity platforms; the shuttle and ISS. These comparisons had never before been investigated.
Understanding proteins is basic to understanding the processes of living things. While we know the chemical formulae of proteins, learning the chemical structure of these macromolecules is more difficult. Mapping the three-dimensional structure of proteins, DNA, ribonucleic acid (RNA), carbohydrates, and viruses provides information concerning their functions and behavior. This knowledge is fundamental to the emerging field of rational drug design, replacing the trial-and-error method of drug development. Microgravity provides a unique environment for growing crystals, an environment that is free of the gravitational properties that can crush the delicate structures of crystals. Currently, several test facilities are used to grow crystals.
The Advanced Protein Crystallization Facility (APCF) can support three crystal-growth methods: liquid-liquid diffusion, vapor diffusion, and dialysis. Liquid-liquid diffusion was not used during Expedition 3. In the vapor diffusion method, a crystal forms in a protein solution as a precipitant draws moisture in a surrounding reservoir. In the dialysis method, salt draws moisture away from the protein solution via a membrane separating the two, forming crystals. ESA has announced that due to potential difficulties with the vapor diffusion method that could cause experiment failure, it will no longer propose the use of this method with the APCF.
APCF-PPG10 was one of eight protein crystal investigations that was conducted in the Advanced Protein Crystallization Facility onboard the ISS during Expedition 3. A video camera monitored PPG10 growth and motion of crystals in the APCF on ISS as well as those being grown back on Earth. Technical problems onboard the ISS resulted in image analysis being available for only the first 40 days of the experiment and a less than 80% coverage of the reactor area.
PPG10 is a collagen protein found in many tissues. This collagen is particularly concentrated in the skin, joints and bones. Research in this area could be beneficial to the diagnosis and treatment of diseases such as osteoporosis. PPG10 was the first and most widely investigated of collagen-like polypeptides. Modeling of its triple helix structure and rod shape has led to a greater understanding of the structure and thermodynamic properties of collagens. Until now, this work was hampered by the relatively poor quality of polypeptide crystals grown on Earth.
The crystals that are grown in microgravity are able to grow larger and better organized than ones grown on Earth. The research that is done on these crystals may further human space exploration efforts by technological and biological advancements developed 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 enough quality or large enough size. Consequently, the growth of high quality macromolecular crystals for diffraction analyses 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 medicines.
Operational Requirements and Protocols
The APCF consisted of a processing chamber and an array of support systems including power and data electronics, thermal control system, and video equipment. The APCF processing chamber accommodated 48 modular reactors. Designed to fit into a single EXPRESS locker, the reactors were activated and deactivated in groups of 12 by electronic motors, allowing groups of experiments to be started at different times during APCF's stay on Station. Ten of these reactors were observed by a high-resolution video camera, which allowed investigators to study crystal growth development. The optical system was mounted on a drive to enable direct observation of a protein chamber, 10 reactors in a sequence, 5 on each side. Five of the reactors were observed with a wide field of view, five with a narrow field. In addition, a Mach-Zender interferometer in the APCF made it possible to observe five of the reactors and to measure and visualize changes in the refractive index as the crystals grew.
The Advanced Protein Crystallization Facility was computer controlled and designed to run automatically, providing undisturbed nucleation. The crew checked the status of the facility by reading the LEDs mounted on the front panel daily. APCF required continuous and auxiliary power from the Station via EXPRESS Rack. Video and computer data was also sent via the Station computer to ground operators.
The APCF reactors were filled in Europe and shipped to the Kennedy Space Center ten days before launch. The reactors were activated after transfer to EXPRESS Rack 1 on ISS. The first processing method, vapor diffusion, allowed crystals to form inside a drop of protein solution. The second processing method, dialysis, separated the protein and salt solutions with a membrane. The facility's processing chamber was maintained at 20 degrees C and temperature data was recorded throughout the mission. Camera images in black and white were digitized and stored on the facility tape recorder. Data electronics recorded and stored other information.
On return to Earth, the protein crystals produced in the APCF were examined by crystallography and computers made the mathematical calculations needed to enable three-dimensional modeling of the proteins structures. Images from the video camera and data from the interferometer enabled investigators to study crystal growth development.
Decadal Survey Recommendations
Information Pending^ back to top
Initial analysis of crystals returned from station support the findings of earlier APCF flights: comparative crystallographic analysis indicates that space-grown crystals are superior in every way to control-group crystals grown on Earth under identical conditions (except the critical space environment). Crystals grown in microgravity generally have improved morphology, larger volume, higher diffraction limit, and lower mosaicity as compared to Earth-grown crystals. The researchers reported that the electron-density maps calculated from diffraction data contained considerably more detail, allowing them to produce more accurate three-dimensional models (Vergara, 2005).
Early published results have come out for crystals of (Pro-Pro Gly)10 (PPG10). PPG10 is a collagen protein found in many tissues. This collagen is particularly concentrated in the skin, joints, and bones. Video that was collected during Expedition 3 showed the small movements within the crystallizing solutions. A direct correlation between crystal motion and acceleration from events on station (such as docking, venting, and crew movement) was determined for the first time. The PPG10 crystals were independently studied by X-ray diffraction in various labs; the best resolution attained for microgravity-grown crystals from ISS was 1.5A, superior to the 1.7A obtained on the ground. The teams of APCF scientists are combining data from previous space flights, the ground, and the station to get the best possible information on protein structures for applications in pharmaceutical and physiological research (Vergara, 2005).
PPG10 crystals grown onboard the ISS were observed to move coherently and followed parallel trajectories which was different from movements observed aboard the shuttle. These movements have been linked to large-scale acceleration events such as the undocking of the shuttle, change in ISS attitude and the venting of water and air. Final distribution of the crystals in solution was strongly affected by this motion. Crystal appearance time and growth rate was comparable in all crystal environments (agarose gel in microgravity, solution on Earth and agarose gel on Earth). These observations suggest that the crystal growth mechanism is kinetically controlled (Vergara, 2002 and Berisio,2002).
Using two other experiment aboard ISS, MAMS (Microgravity Acceleration Measurement System which measured residual gravity on the experiment) and SAMS (Space Acceleration Measurement System which measured acceleration caused by space craft docking and undocking, change in ISS attitude, venting and crew movement), a direct correlation between crystal motion and acceleration was determined for the first time. However, this paper reports that there is no apparent correlation between the resulting crystal motions and crystal quality (Castagnolo, 2002).
Castagnolo D, Piccolo C, Carotenuto L, Vegara A, Zagari A. Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 3. Analysis of residual acceleration-induced motion. Acta Crystallographica Section D: Biological Crystallography. 2003; 59(pt4): 773-776.
Vergara A, Corvino E, Sorrentino G, Piccolo C, Tortora A, Carotenuto L, Mazzarella L, Zagari A. Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 1. Video observation. Acta Crystallographica Section D: Biological Crystallography. 2002; 58: 1690-1694. DOI: 10.1107/S0907444902014269.
Berisio R, Vitagliano L, Vergara A, Sorrentino G, Mazzarella L, Zagari A. Crystallization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 2. Comparison of crystal quality by X-ray diffraction. Acta Crystallographica Section D: Biological Crystallography. 2002; 58: 1695-1699. DOI: 10.1107/S0907444902014427.
Castagnolo D, Piccolo C, Carotenuto L, Vergara A, Zagari A. Crystalization of the collagen-like polypeptide (PPG)10 aboard the International Space Station. 3. Analysis of residual acceleration-induced motion. Acta Crystallographica Section D: Biological Crystallography. 2003; 59(pt4): 773-776. DOI: 10.1107/S0907444903002178.
Ground Based Results Publications
Berisio R, Vitagliano L, Mazzarella L, Zagari A. Crystal structure of the collagen triple helix model [(Pro-Pro-Gly)10]3. Protein Science. 2002 February; 11(2): 262-270. DOI: 10.1110/ps.32602. [Received from PI. related, USML-2 and LMS. ]
Berisio R, Vitagliano L, Mazzarella L, Zagari A. Recent progress on collagen triple helix structure, stability and assembly. Protein and Peptide Letters. 2002 April 1; 9(2): 107-116. DOI: 10.2174/0929866023408922. PMID: 12141907. [Received from PI. related, USML-2 and LMS.]
Lorber B. The crystallization of biological macromolecules under microgravity: a way to more accurate three-dimensional structures?. Biochimica et Biophysica Acta. 2002; 1599(1-2): 1-8. PMID: 12479400.
NASA Fact Sheet
Selected PPG10 crystals in reactor number 410 containing a gel-free solution. The photo was taken at Kennedy Space Center one day after landing. Image courtesy of NASA.
+ View Larger Image
PPG10 crystals grown in microgravity, in reactor number 612 containing agarose gel. Image courtesy of NASA.
+ View Larger Image