Advanced Protein Crystallization Facility - Crystallization of Human Low Density Lipoprotein (LDL) Subfractions in Microgravity (APCF-Lipoprotein) - 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
Lipoproteins are lipids – organically soluble but water-insoluble fatty compounds – bound to one or more proteins. In human blood, lipoproteins transport water-insoluble fats, including cholesterol, which is a leading factor in heart and arterial disease. While this investigation successfully produced high-quality lipoprotein crystals, those crystals did not achieve the expected level of resolution seen in others produced on-board the International Space Station. Some of the characteristics were improved over Earth-grown crystals, however, and these results may help researchers learn more about cholesterol-related diseases. Experiment Details
Manfred W. Baumstark, M.D., University of Frieburg, Frieburg, Germany
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.
- APCF-Lipoprotein was designed to produce lipoprotein crystals of significantly better resolution than those grown on Earth. Lipoproteins are a complex of one or more lipids (fatty compounds that are characteristically insoluble in water but readily soluble in organic solvents) bound to one or more proteins. In humans, lipoproteins transport water-insoluble fats in the blood.
- Cholesterol, a leading factor in heart disease and hardening of the arteries, is carried through the bloodstream by lipoproteins. Obtaining and studying high-quality lipoprotein crystals will aid researchers in learning more about the cause and prevention of cholesterol related diseases.
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-Lipoprotein was one of eight protein crystal investigations that was conducted in the Advanced Protein Crystallization Facility onboard the ISS during Expedition 3. Lipoproteins are a complex of one or more lipids bound to one or more proteins that transport water-insoluble fats in the blood. They are classified by their density: very-low-density lipoproteins (VLDLs), low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs). Fatty acids and steroids such as LDL cholesterol (a leading cause of heart disease and hardening of the arteries) are transported through the bloodstream by lipoproteins. Studying lipoprotein structure in high-quality crystals will help researchers learn more about the cause and prevention of cholesterol related diseases.
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 drugs.
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.
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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).
The APCF hardware performed well during ISS Expedition 3 with very few anomalies. APCF-Camelids, APCF-Crystal Quality, APCF-Crystal Growth, APCF Octarellins and APCF-PPG10 all produced excellent quality crystals that had better resolution and other optical properties than those grown on Earth. APCF-Lipoprotein successfully produced crystals but they did not achieve the expected level of resolution. APCF-Rhodopsin had slight technical problems that prevented the formation of suitable crystals.
Well-diffracting crystals prepared in space have two purposes: understanding the gravity-dependent phenomena (such as nucleation and growth mechanisms) and structural determination. Each new high-resolution structure may become the start of a cascade of investigations to unravel the complexity of the cellular events like growth, division, differentiation, communication, motility, death and their role in the development of multicellular organisms. This may accelerate the structure-based design and redesign of drugs targeting pathogens, diseases and degenerative cellular processes as well as of protein and nucleic acid leading to custom enzymes, ribozymes or inhibitors to treat diseases (Lober, 2002).
Although APCF-Lipoprotein successfully produced crystals, the main objective of achieving a significant improvement in resolution (compared to Earth-grown crystals) was not accomplished. However, there was some indication that parameters like mosaicity and the signal/noise ratio were in fact improved (Baumstark, M. "APCF-ISS Expedition 3." 11 November 2005. Personal. firstname.lastname@example.org 28 November 2005).
Vergara A, Lorber B, Sauter C, Giege R, Zagari A. Lessons from crystals grown in the Advanced Protein Crystallisation Facility for conventional crystallization applied to structural biology. Biophysical Chemistry. 2005; 118(2-3): 102-112. DOI: 10.1016/j.bpc.2005.06.014. PMID: 16150532.
Ground Based Results Publications
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
NASA Image: ISS003E8171 - The Advanced Protein Crystal Facility located in EXPRESS Rack 1 onboard ISS Expedition 3. Two other experiments, MAMS and SAMS-II, are installed below it.
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