Comparison of the three-dimensional structures of a human Bence-Jones dimer crystallized on Earth and aboard US Space Shuttle Mission STS-95

Crystals of a human (Sea) Bence-Jones dimer were produced in a capillary by vapor diffusion under microgravity conditions in the 9 day US Space Shuttle Mission STS-95. In comparison to ground-based experiments, nucleation was facile and spontaneous in space. Appearance of a very large (8 x 1.6 x 1.0 mm) crystal in a short time period is a strong endorsement for the use of microgravity to produce crystals sufficiently large for neutron diffraction studies. The Sea dimer crystallized in the orthorhombic space group P2(1)2(1)2(1), with a = 48.9 A, b = 85.2 A, and c = 114.0 A. The crystals grown in microgravity exhibited significantly lower mosaicities than those of ground-based crystals and the X-ray diffraction data had a lower overall B factor. Three-dimensional structures determined by X-ray analysis at two temperatures (100 and 293 K) were indistinguishable from those obtained from ground-based crystals. However, both the crystallographic R factor and the free R factor were slightly lower in the models derived from crystals produced in microgravity. The major difference between the two crystal growth systems is a lack of convection and sedimentation in a microgravity environment. This environment resulted in the growth of much larger, higher-quality crystals of the Sea Bence-Jones protein. Structurally, heretofore unrecognized grooves on the external surfaces of the Sea and other immunoglobulin-derived fragments are regular features and may offer supplementary binding regions for super antigens and other elongated ligands in the bloodstream and perivascular tissues.     

Structural study on hen egg-white lysozyme crystals grown in gravity and microgravity

Ｐｒｏｔｅｉｎｃｒｙｓｔａｌｇｒｏｗｔｈｈａｓｂｅｅｎｏｆｇｒｅａｔｉｍｐｏｒｔａｎｃｅｉｎｅｘｐｌｏｉｔａｔｉｏｎｏｆｓｔｒｕｃｔｕｒｅｓｏｆｂｉｏｌｏｇｉｃａｌｍａｃｒｏｍｏｌｅｃｕｌｅｓ.Ｔｈｅｍｉｃｒｏｇｒａｖｉｔｙｅｎｖｉｒｏｎｍｅｎｔｉｎｓｐａｃｅｉｓａｎｉｄｅａｌｐｌａｃｅｔｏｓｔｕｄｙｔｈｅｃｏｍｐｌｉｃａｔｅｄｐｒｏｔｅｉｎｃｒｙｓｔａｌｌｉｚａｔｉｏｎｐｒｏｃｅｓｓａｎｄｔｏｇｒｏｗｇｏｏｄｑｕａｌｉｔｙｐｒｏｔｅｉｎｃｒｙｓｔａｌｓ.Ｓｉｎｃｅｔｈｅ1980ｓ,ｎｏｔｉｃｅａｂｌ…     

微重力下天花粉蛋白晶体生长初探

报导了国内首次在微重力下进行蛋白质晶体生长的试验.与地面对照实验所生长的晶体相比较,在空间生长的多数天花粉蛋白晶体具有较完整的外形,初步展示了微重力条件对蛋白质晶体生长所具有的优越性.     

Protein crystallization in microgravity.

A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.     

Cell science and protein crystal growth research for the International Space Station

The recent National Research Council report, Future Biotechnology Research on the International Space Station, evaluates NASA's plans for research in cell science and protein crystal growth to be conducted on the International Space Station. This report concludes that the NASA biotechnology programs have the potential to significantly impact relevant scientific fields and to increase understanding and insight into fundamental biological issues. In order to realize the potential impacts, NASA must focus its research programs by selecting specific questions related to gravitational forces' role in cell behavior and by using the microgravity environment as a tool to determine the structure of macromolecules with important biological implications. Given the time and volume constraints associated with space-based experiments, instrumentation to be used on the space station must be designed to maximize the productivity of researchers, and NASA's recruitment of investigators and support for space station experiments should aim to encourage and facilitate cutting-edge research.     

The second space experiment of protein crystallization with domestic facilities

The second experiment of protein crystallization was performed on domestic re-entry satelliteFSW-2 in 1994-07. The results are superior to the ones of the first mission in 1992: 9 of 10 different proteins were crystallized in space, and 70% of the total 48 samples yielded single crystals. Besides hen egg-white lysozyme which grew high-quality crystals on the first mission, an acidic phospholipase A2(aPLA2) from snake venom and hemoglobin from Anser Indicus produced good-quality crystals suitable for X-ray diffraction analyses. The positive effect of microgravity on protein crystal growth is verified again at this time.     

微重力环境影响植物生长发育的研究进展

微重力是最独特的空间环境条件之一,研究微重力对不同植物种类以及不同植物部位的影响是空间生物学的重要内容之一,对于建立生物再生式生命保障系统意义重大。生物再生式生命保障系统是未来开展长期载人空间活动的核心技术,其优势在于能在一个密闭的系统内持续再生氧气,水和食物等高等动物生活必需品,植物部件是生物再生式生命保障系统的重要组成部分。了解和掌握微重力对植物生长发育的影响,有助于采取有效的作业制度确保其正常生长发育和繁殖,是成功建立生物再生式生命保障系统的首要关键。该文就植物在空间探索中的地位和作用,地面模拟微重力的装置以及国内外有关微重力对植物的影响做一综述。现有的研究结果包括,未来长期的载人航天任务需要植物通过光合作用为生物再生式生命保障系统提供部分动物营养、洁净水以及清除系统中的固体废物和二氧化碳;三维随机回旋装置是目前地面上模拟微重力效应的主要装置之一,尤其适用于植物材料的长期模拟微重力处理;国内外有关微重力对植物影响的报道生理生化水平多集中在植物的生长发育和生理反应,比如表型变化或者与重力相关的激素或者钙离子的再分配,细胞或亚细胞水平主要有细胞壁、线粒体、叶绿体以及细胞骨架等,基因和蛋白质表达水平的研究对象主要为拟南芥。由于实验方法和材料之间的差异,微重力对不同植物或者植物不同部位在各个水平的影响效果并不一致,未来需要开展更多的相关研究工作。     

Protein crystal growth on board Shenzhou 3: a concerted effort improves crystal diffraction quality and facilitates structure determination

The crystallization of 16 proteins was carried out using 60 wells on board Shenzhou 3 in 2002. Although the mission was only 7 days, careful and concerted planning at all stages made it possible to obtain crystals of improved quality compared to their ground controls for some of the proteins. Significantly improved resolutions were obtained from diffracted crystals of 4 proteins. A complete data set from a space crystal of the PEP carboxykinase yielded significantly higher resolution (1.46A vs. 1.87A), I/sigma (22.4 vs. 15.5), and a lower average temperature factor (29.2A(2) vs. 42.9A(2)) than the best ground-based control crystal. The 3-D structure of the enzyme is well improved with significant ligand density. It has been postulated that the reduced convection and absence of macromolecule sedimentation under microgravity have advantages/benefits for protein crystal growth. Improvements in experimental design for protein crystal growth in microgravity are ongoing.     

Protein and virus crystal growth on international microgravity laboratory-2.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.     

Lessons from crystals grown in the Advanced Protein Crystallisation Facility for conventional crystallisation applied to structural biology

The crystallographic quality of protein crystals that were grown in microgravity has been compared to that of crystals that were grown in parallel on earth gravity under otherwise identical conditions. A goal of this comparison was to assess if a more accurate 3D-structure can be derived from crystallographic analysis of the former crystals. Therefore, the properties of crystals prepared with the Advanced Protein Crystallisation Facility (APCF) on earth and in orbit during the last decade were evaluated. A statistical analysis reveals that about half of the crystals produced under microgravity had a superior X-ray diffraction limit with respect of terrestrial controls. Eleven protein structures could be determined at previously unachieved resolutions using crystals obtained in the APCF. Microgravity induced features of the most relevant structures are reported. A second goal of this study was to identify the cause of the crystal quality enhancement useful for structure determination. No correlations between the effect of microgravity and other system-dependent parameters, such as isoelectric point or crystal solvent content, were found except the reduced convection during the crystallisation process. Thus, crystal growth under diffusive regime appears to be the key parameter explaining the beneficial effect of microgravity on crystal quality. The mimicry of these effects on earth in gels or in capillary tubes is discussed and the practical consequences for structural biology highlighted.     

Influence of microgravity on crystal formation in biomineralization.

Biomineralized tissues are widespread in animals. They are essential elements in skeletons and in statocysts. The function of both can only be understood with respect to gravitational force, which has always been present. Therefore, it is not astonishing to identify microgravity as a factor influencing biomineralization, normally resulting in the reduction of biomineralized materials. All known biominerals are composite materials, in which the organic matrix and the inorganic materials, organized in crystals, interact. If, during remodeling and turnover processes under microgravity, a defective organization of these crystals occurs, a reduction in biomineralized materials could be the result. To understand the influence of microgravity on the formation of biocrystals, we studied the shell-building process of the snail Biomphalaria glabrata as a model system. We show that, under microgravity (space shuttle flights STS-89 and STS-90), shell material is built in a regular way in both adult snails and snail embryos during the beginning of shell development. Microgravity does not influence crystal formation. Because gravity has constantly influenced evolution, the organization of biominerals with densities near 3 must have gained independence from gravitational forces, possibly early in evolution.     

微重力条件下细胞培养和组织工程研究进展

随着空间生命科学研究的发展,人们将细胞、组织培养技术与微重力环境相结合产生了组织工程研究的一个新领域——微重力组织工程。模拟微重力条件下细胞培养和组织构建研究表明,微重力环境有利于细胞的三维生长,形成具有功能的组织样结构,培养后的三维组织无论从形态上还是基因表达上都更接近于正常的机体组织。这种微重力对细胞的作用效应,将可能为未来组织工程和再生医学研究提供一条新途径。该文概述了近十年来国内外微重力组织工程相关研究的最新进展。     

Gravitational biology and space life sciences: Current status and implications for the Indian space programme

This paper is an introduction to gravitational and space life sciences and a summary of key achievements in the field. Current global research is focused on understanding the effects of gravity/microgravity on microbes, cells, plants, animals and humans. It is now established that many plants and animals can progress through several generations in microgravity. Astrobiology is emerging as an exciting field promoting research in biospherics and fabrication of controlled environmental life support systems. India is one of the 14-nation International Space Exploration Coordination Group (2007) that hopes that someday humans may live and work on other planets within the Solar System. The vision statement of the Indian Space Research Organization (ISRO) includes planetary exploration and human spaceflight. While a leader in several fields of space science, India is yet to initiate serious research in gravitational and life sciences. Suggestions are made here for establishing a full-fledged Indian space life sciences programme.     

Continuous flow electrophoretic separation of proteins and cells from mammalian tissues

A new continuous flow electrophoretic separator for cells and macromolecules was built and tested in laboratory experiments and in the microgravity environment of space flight. Buffer flows upward in a 120-cm long flow chamber, which is 6 cm wide X 1.5 mm thick in the laboratory version and 16 cm wide X 3.0 mm thick in the microgravity version. Electrophoretic subpopulations are collected in 197 fractions spanning 16 cm at the upper end of the chamber. The electrode buffer is recirculated through front and back cooling chambers, which are also electrode chambers. Ovalbumin and rat serum albumin were used as test proteins in resolution and throughout tests; resolution of these two proteins at 25% total w/v concentration in microgravity was the same as that found at 0.2% w/v concentration in the laboratory. Band spreading caused by Poiseuille flow and conductance gaps was evaluated using polystyrene microspheres in microgravity, and these phenomena were quantitatively the same in microgravity as in the laboratory. Rat anterior pituitary cells were separated into subpopulations enriched with cells that secrete specific hormones; growth-hormone-secreting cells were found to have high electrophoretic mobility, whereas prolactin-secreting cells were found to have low electrophoretic mobility. Cultured human embryonic kidney cells were separated into several electrophoretic subfractions that produced different plasminogen activators; a medium-high-mobility subpopulation and a medium-low-mobility subpopulation each produced a different molecular form of urokinase, whereas a high- and an intermediate-mobility subpopulation produced tissue plasminogen activator. Canine pancreatic islets of Langerhans cells were separated into subpopulations, which, after reaggregation into pseudoislets, were found to be enriched with cells that secrete specific hormones; insulin-secreting beta cells were found in lowest mobility fractions, whereas glucagon-secreting alpha cells were found in the highest mobility fractions. Results of particle electrophoresis experiments were comparable in microgravity and in the laboratory, since cell densities that overloaded the carrier buffer (resulting in zone sedimentation) were avoided, and a 500-fold increase in protein throughput was achieved without compromising resolution in microgravity.     

A Microfluidic,High Throughput Protein Crystal Growth Method for Microgravity

The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10³ cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.     

Space radiation effects and microgravity

Humans in space are exposed both to space radiation and microgravity. The question whether radiation effects are modified by microgravity is an important aspect in risk estimation. No interaction is expected at the molecular level since the influence of gravity is much smaller than that of thermal motion. Influences might be expected, however, at the cellular and organ level. For example, changes in immune competence could modify the development of radiogenic cancers. There are no data so far in this area. The problem of whether intracellular repair of radiation-induced DNA lesions is changed under microgravity conditions was recently addressed in a number of space experiments. The results are reviewed; they show that repair processes are not modified by microgravity.     

Secondary metabolism in simulated microgravity and space flight

Space flight experiments have suggested that microgravity can affect cellular processes in microorganisms. To simulate the microgravity environment on earth, several models have been developed and applied to examine the effect of microgravity on secondary metabolism. In this paper, studies of effects of space flight on secondary metabolism are exemplified and reviewed along with the advantages and disadvantages of the current models used for simulating microgravity. This discussion is both signi?cant and timely to researchers considering the use of simulated microgravity or space flight to explore effects of weightlessness on secondary metabolism.     

Space biotechnology: current and future perspectives

The potential for obtaining enhanced purities and for achieving greater homogeneity of materials in microgravity first attracted biotechnologists to space bioprocessing. This is but one of the benefits of microgravity. This review discusses the unique opportunities of space biotechnology and the diverse means to achieve microgravity conditions.     

Effects of simulated microgravity on nitric oxide level in cardiac myocytes and its mechanism

The depression of cardiac contractility induced by space microgravity is an important issue of aerospace medicine research, while its precise mechanism is still unknown. In the present study, we explored effects of simulated microgravity on nitric oxide (NO) level, inducible nitric oxide synthase (iNOS) expression and related regulative mechanism using electron spin resonance (ESR) spectroscopy, immunocytochemistry and in situ hybridization. We found a remarkable increase of NO level and up-regulation of iNOS and iNOS mRNA expression in rat cardiac myocytes under simulated microgravity. Staurosporine (a nonselective protein kinase inhibitor), calphostin C (a selective protein kinase C inhibitor), partially inhibited the effect of simulated microgravity. Thus regulative effect of simulated microgravity on iNOS expression is mediated at least partially via activation of protein kinase C. These results indicate that NO system in cardiac myocytes is sensitive to simulated microgravity and may play an important role in the depression of cardiac contractility induced by simulated microgravity.     

The crystallization of biological macromolecules under microgravity: a way to more accurate three-dimensional structures?

The crystallization of proteins and other biological particles (including nucleic acids, nucleo-protein complexes and large assemblies such as nucleosomes, ribosomal subunits or viruses) in a microgravity environment can produce crystals having lesser defects than crystals prepared under normal gravity on earth. Such microgravity-grown crystals can diffract X-rays to a higher resolution and have a lower mosaic spread. The inferred electron density maps can be richer in details owing to which more accurate three-dimensional structure models can be built. Major results reported in this field of research are reviewed. Novel ones obtained with the Advanced Protein Crystallization Facility are presented. For structural biology, practical applications and implications associated with crystallization and crystallography onboard the International Space Station are discussed.