Results of Space Experiments

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It is estimated that in about 20% of the different proteins and other biological macromolecular assemblies that have been studied in space, the resolution of the crystallographic analysis was better than that of the best ground-based results available at the time. The proteins whose resolution improved in space range from well-understood test cases, such as lysozyme, to proteins that present significant challenges for contemporary structural biology, such as the EcoRI-DNA complex, the nucleosome core particle, and the epidermal growth factor receptor. Enhanced resolution has also been obtained for proteins of importance for drug design, including the HIV protease complexes with lead compounds, and the influenza neuraminidase. Improvements in resolution of crystals of canavalin, Satellite Tobacco Mosaic Virus (STMV), and insulin in microgravity were also substantial.

For example, the analysis of tetragonal lysozyme crystals grown on two Space Shuttle missions showed that the mosaicity of the crystals was improved by factors of 3 or 4 over that observed for lysozyme crystals grown on Earth (Snell et al. 1995) (Figure 8-06). This is a very significant improvement because it can allow the measurement of very weak reflections that would otherwise be too broad to be observed over background. Although crystals of lysozyme with very low mosaicity can occasionally be obtained on Earth, only about 1 in 40 of them have properties comparable to those of the crystals grown in space.

Figure 8-06. A lysozyme crystal grown in orbit. Photo courtesy of NASA.

Another example is STMV, a small icosahedral plant virus consisting of a protein shell made up of 60 identical protein subunits of molecular weight 14,000, which has been studied extensively on Earth (Figure 8-07). Its crystallization in microgravity was investigated during two Space Shuttle missions in 1992 and 1994. Using a liquid-liquid diffusion technique with careful temperature control, crystals of STMV obtained in space were about 10 times larger in volume than the largest crystals of STMV previously grown in ground-based laboratories (Day and McPherson 1992). In contrast to the crystals grown on Earth, the crystals grown in microgravity were visually perfect, with no striations or clumping of crystals. Furthermore, the X-ray diffraction data obtained from the space-grown crystals was of a much higher quality than the best data available at that time from ground-based crystals. STMV also crystallizes on Earth in a cubic crystal form that diffracts poorly; at the time of the 1994 shuttle flight, the best available ground results gave

Figure 8-07. Crystal of the Satellite Tobacco Mosaic Virus (STMV) and computergenerated model of its protein structure.

only about 6 A resolution (Koszelak et al. 1995). Cubic crystals of STMV obtained on board the Space Shuttle were 30 times larger than those obtained on Earth. These crystals diffracted X-rays to 4 A resolution, a significant improvement over the ground-based crystals.

Figure 8-07. Crystal of the Satellite Tobacco Mosaic Virus (STMV) and computergenerated model of its protein structure.

As with STMV, large crystals of canavalin, a plant storage protein, were obtained in space (Koszelak et al. 1995). Visually perfect crystals of canavalin were obtained in large numbers (Figure 8-08), with significantly better diffraction properties than those of crystals grown on Earth. The diffraction limit was extended from 2.7 A (Earth) to nearly 2.2 A (space), and the total number of useful X-ray measurements essentially doubled.

Figure 8-08. Hexagonal ctystals of canavalin grown in the Protein Crystal Growth (PCG) facility on board the Mir station. Photo courtesy of NASA.

Hexagonal Protein Crystals

Figure 8-08. Hexagonal ctystals of canavalin grown in the Protein Crystal Growth (PCG) facility on board the Mir station. Photo courtesy of NASA.

Another example is with insulin, a hormone released by the pancreas in response to increased levels of sugar in the blood. Insulin aggregates to form hexamers, which undergo a change from one three-dimensional arrangement of atoms and bonds to another. The switching between the two states is altered by the presence of particular ions and organic molecules, and there is interest in identifying additives that would stabilize one state, which would lead to insulin preparations with greater stability. To this end, highresolution crystallographic analyses of insulin have being carried out. Crystals of human insulin grown in space were larger and free of imperfections compared with crystals grown on Earth (Figure 8-09). A resolution of 0.9 A has been obtained using synchrotron X-ray radiation on T6 insulin crystallized on board the Space Shuttle, whereas data to 1.9 A resolution were obtained using the crystals grown on Earth and a laboratory X-ray source (Smith et al. 1996). This ultrahigh resolution data is allowing very detailed analysis of the molecular structure, including the study of electronic distributions within the protein molecule. This data will add information for the development of new therapeutic insulin treatments for the control of diabetes. Such treatments would greatly improve the quality of life of people on insulin therapy by reducing the number of injections they require and by reducing the cost of treatment.

Figure 8-09. Space-grown (left) and Earth-grown (right) insulin crystals. Photo courtesy of NASA.

The four cases described above provide the most convincing data currently available on the benefits of growing protein crystals in microgravity. Several dozens of other experiments produced space-grown crystals with improved resolution, e.g:

• Factor D protein crystals - led to development of a drug that may aid patients recovering from open heart surgery.

• Antithrombin - a protein which controls blood coagulation in human plasma, which has important implications for medicine.

• HIV protease/inhibitor complex - may have applications for designing new drugs for AIDS therapies.

• Influenza protein crystals and neuraminidase - a target for the treatment and prevention of the flu.

• Proteins associated with Chagas' disease - a debilitating and deadly disease that affects more than 20 million people in Latin America and parts of the United States.

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