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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
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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
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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
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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
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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.
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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
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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
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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.
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