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Toward a Microgravity Research Strategy (Appendix A) Toward a Microgravity Research Strategy A Biological Sciences Biotechnology encompasses the sciences based on molecular biology and the engineering and technological developments needed to convert discoveries into useful products. The biotechnology field is one of the most rapidly developing and widely ranging areas of research, Typically, the biological research considered for microgravity is divided into the study of isolated biomacromolecules (proteins and nucleic acids) and their assemblies, organelles, and cells in controlled fluid and chemical environments. According to the NASA DWG on biological sciences, the microgravity environment offers advantages for (1) examining the physical chemistry of biomolecular structures and their interaction, most specifically by protein crystallography; (2) using separation processes to provide improved sensitivity in preparative and analytical techniques; and (3) studying cells and cell cultures. To date, there has been only slight progress in the application of REPORT MENU microgravity sciences to any of these thrusts in the biological sciences. Hence, NOTICE most of the discussion that follows is based on prospects and not on completed MEMBERSHIP research. SUMMARY CHAPTER 1 CHAPTER 2 CHAPTER 3 STATUS CHAPTER 4 APPENDIX A APPENDIX B Protein Crystallography APPENDIX C APPENDIX D Protein crystallography is the general name given to determination of the APPENDIX E detailed, three-dimensional structure of biological macromolecules, including APPENDIX F protein, DNA, and RNA, by using x-ray crystallographic techniques. The determination of accurate macromolecular structures is absolutely necessary for establishing the molecular mechanisms of biological reactions, for rational drug design (in which a molecule is designed to bind to a specific target protein), and for the design of proteins and nucleic acids with new activities and functions. During the past decade, the methods of protein crystallography have been made faster and more accurate through the use of improved data collection methodologies and more powerful computers. Now, virtually all proteins and file:///C|/SSB_old_web/cmgr92appenda.htm (1 of 7) [6/18/2004 11:09:48 AM]

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Toward a Microgravity Research Strategy (Appendix A) nucleic acids can be made in sufficiently large quantities for crystallographic analysis using the methodologies of cloning genes or by direct chemical synthesis. This means that any biological macromolecule for which there is a gene sequence can, in principle, become a subject for study by protein crystallography. This will become increasingly important as large numbers of sequences for proteins of unknown structure and function become available through the efforts of the Human Genome Project. The amino acid sequence of a protein of either known or unknown function cannot be interpreted usefully in the absence of a three-dimensional structure. Determination of crystal structure by protein crystallography requires well- ordered, single crystals whose minimum dimension is 0.2 to 0.4 mm and whose maximum dimension need not be larger than 1 mm. An optimal crystal size for most biological macromolecules would be about 1 mm in all three dimensions. The intensity of the diffraction pattern increases as the cube of the linear dimension increases up to this optimal size. Thus, crystals much smaller than 0.1 mm in maximum dimension are not useful for crystal structure determination because the x-ray diffraction pattern that results from such a crystal is too weak to be measured accurately. The accuracy of the resulting coordinates derived from determining a crystal structure is directly related to the resolution to which the crystals diffract. The resolution is quoted in terms of the minimum spacing in angstroms between Bragg planes that can be resolved. The ratio of measurable data to refinable parameter increases as the inverse cube of the resolution of the measurable diffraction pattern; that is, there are eight times more data at 2-Å resolution than at 4-Å resolution. Proteins whose structures are refined at 2 Å have coordinate errors of 0.2 to 0.3 Å in favorable cases. Structures determined at higher resolutions have smaller errors in atomic coordinates, while those determined at lower resolutions can have significantly larger errors. During the past 10 to 15 years, new techniques of crystallization, particularly microcrystallization, have greatly improved the speed, yield, and quality of crystals grown in various laboratories around the world. In turn, these improvements in crystallization methodologies have increased immensely the usefulness and impact of protein crystallography in the biological community in general. It is very probable that any general improvement in the ability to grow suitable crystals of macromolecules will have a major impact on crystallography's usefulness to the biomedical community. Separation Processes In many instances, progress in the biological sciences and in biotechnology is limited by the ability to separate a myriad of proteins, nucleic acids, and associated complexes created by modern genetic engineering either as natural mixtures in blood serum or from manufactured mixtures, such as cell culture media, hybridoma cultures, or synthetic solutions. Many of the separation file:///C|/SSB_old_web/cmgr92appenda.htm (2 of 7) [6/18/2004 11:09:48 AM]

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Toward a Microgravity Research Strategy (Appendix A) processes currently in use are affected deleteriously by gravity. For example, buoyancy-driven convection disturbs sedimentation processes. To counterbalance the action of gravity, the most common method used for analytical separations is electrophoresis carried out in water-based gels. The gel serves to limit hydrodynamic convection because it causes muchreduced separation rates. A major microgravity research program has focused on the study of liquid- based electrohydrodynamics, which has been shown to limit the scaleup in space of electrophoresis experiments for a given separation efficiency. Other bioseparation methods also have received attention, for example, isoelectric focusing used in protein analysis. Again, the electrohydrodynamic effects have been found to be important, Other methods include phase separation based on chemical partitioning. The efficiency of these methods hinges on the ability to maintain a very dispersed phase and is influenced strongly by sedimentation. Cells and Cell Cultures Current biological research includes the formation of ordered biocompatible materials, genetic manipulation, cell fusion, and the regulation of cell growth and differentiation. One of the most challenging aspects of these problems is the manipulation of fragile mammalian and plant cells, which are very sensitive to hydrodynamic shear forces and to interactions with container walls, and have complex metabolic requirements. The microgravity environment has potential for the conduct of cell science research. Ground-based research is under way to characterize the fluid dynamic environment of cell culture systems and includes studies of the hydrodynamics of cell suspensions during separation. The influence of hydrodynamic, cell-cell, and cell-container interactions on setting upper limits of cell densities needs to be determined. New devices for cell cultures are designed to provide low shear rates and minimal sedimentation. To date, the viability of these concepts is unproven. Finally, the fusion of cells to produce viable hybrid cells is another technology that may benefit from microgravity. Cell fusion requires the interaction of two cell types and the annealing of the cell membrane to yield ii single cell: usually, this is a low-frequency event in current experiments on Earth. The cells generally have different densities, and so sedimentation tends to separate the cells during fusion. It has been hypothesized that a microgravity environment would alleviate these tendencies and lead to higher fusion rates. Support for this hypothesis has come from experiments on the German Spacelab and on TEXUS sounding rocket flights; both exhibited fusion in microgravity of yeast cells. file:///C|/SSB_old_web/cmgr92appenda.htm (3 of 7) [6/18/2004 11:09:48 AM]

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Toward a Microgravity Research Strategy (Appendix A) MAJOR RESEARCH ACCOMPLISHMENTS Use of the microgravity environment is just beginning to increase our understanding of the biological sciences and to enable us to develop innovative biotechnological processes that can exploit microgravity. Several research projects have resulted in valuable findings in microgravity crystallization and protein separations. Thus far, the NASA-sponsored microgravity crystallization efforts have had two major components. One is a ground-based, systematic examination of the principles and methodologies of protein crystallization. Although these studies are still in their infancy, they show considerable promise and constitute one of a relatively small number of significant systematic approaches to protein crystallization being carried out in the world. The second major effort has been in the growth of protein crystals in space. In addition to NASA's efforts in space, experiments are being carried out by the Europeans and the former Soviets. While the number of experimental examples has been limited, the general conclusion drawn from these crystallization experiments in space is that it is possible to grow larger and morphologically better protein crystals that diffract to higher resolution, in at least some cases. In a recent NASA experiment, crystals of three proteins grew large enough to be examined by x-ray diffraction, and, in each case, the crystals diffracted to higher resolution than the best Earthgrown crystals. In these examples, the amount of measurable data seems to have increased by about a factor of two. In about 40 percent of the crystallization experiments, no crystals at all were obtained, and in another 30 percent, the crystals were too small for x-ray analysis. This is attributed, probably correctly, to the crystallization conditions being suboptimal in the specific microgravity environment used and to the small number of separate crystallization experiments done in the case of each protein. In another program, the potential for achieving high precision in electrophoretic separations has been addressed in microgravity experiments. Separations carried out on Earth and in space, coupled with extensive theoretical analysis, have shown the important role of electrohydrodynamic forces in determining the efficiency of separation; the convective movement of charged molecules in an electric field leads to significant convective mixing in large-gap electrophoresis devices in space and severely degrades the quality of the separation. These effects are masked on Earth by the smallgap devices that must be used to prevent convection due to density gradients. This research exemplifies the role of microgravity experiments in determining the ultimate limits of various separation technologies for veryhigh-value-added materials. RESEARCH PROSPECTS AND OPPORTUNITIES file:///C|/SSB_old_web/cmgr92appenda.htm (4 of 7) [6/18/2004 11:09:48 AM]

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Toward a Microgravity Research Strategy (Appendix A) Imaginative research should be supported in both science and engineering applications. Research should be sponsored in cell science, bioseparation, and protein crystallization. The last topic has perhaps the most potential for an immediate impact on ground-based research. Protein crystal growth is one of the most promising possibilities. An important overall goal of both ground-based and microgravity efforts should be to find factors that exist in crystallization under microgravity conditions in space that are important in yielding the larger, better-formed crystals that diffract to higher resolution and to try to the extent possible to duplicate those conditions on Earth. This would require additional studies of the crystallization process in ground- based laboratories. While the present work on mechanisms of protein crystallization being conducted at the University of Alabama's Center for the Commercial Development of Space is well regarded, an incremental expansion of such fundamental studies would be done most effectively through competitive research grants that could be made available to any of the crystallographic laboratories in the United States. Obtaining the active participation of a larger number of protein crystallographic laboratories in this country on this fundamental aspect of the field of protein crystallography would be most likely to yield important results. The present plans for experiments that are to be performed in space to study the crystallization process and to vary the conditions of crystallization should be continued and expanded. Only by monitoring the crystallization process in space will it be possible to assess the ways in which microgravity and its effects on transport favorably affect the crystallization process. If this could be coupled with the ability to vary crystallization conditions while in space, it might be possible to increase greatly the yield of crystallographically suitable crystals obtained in space. If the reliability of obtaining large, crystallographically suitable crystals in space could be increased considerably by on-board assessment, the method could become more generally feasible and useful to ongoing crystallographic studies. In general, only a small number of large crystals is required to obtain a high-resolution diffraction data set. Most of the crystals required to establish the protein structure could initially be Earth-grown crystals. The use of space-grown crystals to improve the accuracy of structures found using Earth-grown crystals could prove to be a more cost-effective way to use this limited resource. It has not been demonstrated that the growth of crystals in space is the fastest or most cost-effective way of obtaining large crystals from small ones, although the on- board examination of crystal growth could change that conclusion. Up to the present, the rationale for putting x-ray data collection equipment into .space is less convincing. Building the capacity to analyze crystals in space would be extremely expensive, and it is not at all obvious that the general benefit would be commensurate with the very high cost. The reasons for evaluating crystals in space include the speed with which the results of crystallization could be evaluated, the ability to analyze a small number of crystals that might not be file:///C|/SSB_old_web/cmgr92appenda.htm (5 of 7) [6/18/2004 11:09:48 AM]

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Toward a Microgravity Research Strategy (Appendix A) stable for the period of time required to return them to Earth, and the small possibility that some crystals might have their crystallinity distorted by gravity. The promise of protein crystallography and the potential usefulness of microgravity in producing protein crystals of superior quality should not provide any part of the justification for building a space station. Growing crystals of superior quality in space is not close, nor is it likely to become close, to being cost-effective. All proteins and nucleic acids are highly polymorphic in their ability to form crystals. In other words, these biological macromolecules can be crystallized in many different crystal forms under different crystallization conditions; the presence or absence of gravity is merely one of many variables that can be explored in the pursuit of better crystals. Different crystal forms of the same protein frequently differ dramatically in the resolution to which they diffract x rays—from 20-Å to 2-Å resolution. It currently is (and is likely to remain) faster and very much less expensive to obtain superior-quality crystals by changing the form of a crystal as a result of varying crystallization conditions on the ground, rather than by improving the' form of an existing crystal through growing it in space. While improvements in separation processes continue to be important to the study of biological systems, progress in ground-based separations has proceeded at a great pace and probably will have an overwhelmingly greater impact on biomedical research than separation methods in space will have. file:///C|/SSB_old_web/cmgr92appenda.htm (6 of 7) [6/18/2004 11:09:48 AM]