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Protein crystal growth experiments have been performed on fourteen space shuttle missions between April 1985 and June 1992. These space shuttle missions have been used to grow crystals of a variety of proteins using vapour diffusion, liquid diffusion, and temperature induced crystallization techniques. The United States Microgravity Laboratory-1 mission (june 25-July 9, 1992) was a space lab mission dedicated to experiments involved in materials processing. New protein crystal growth hardware was developed to allow in-orbit examination of initial crystal growth results, the knowledge from which was used on subsequent days to prepare new crystal growth experiments. The hardware developed specifically for the USML-1 mission is discussed along with preliminary experimental results.

Two different protein crystallizations, namely ,the free Fab fragment of the Je142 monoclonal antibody and the complex of Fab fragment/HPr with antigen, were performed aboard the Discovery Space Shuttle flights and the Mir space station, respectively. Medium sized crystals of the Je142 Fab fragment were obtained. The Je142 Fab fragment/Hpr complex produced two medium-sized crystals after two months aboard the Mir space station. Microgravity was found to eliminate the tendency of these crystals to form clusters.

Proteins (enzymes, hormones, immunoglobulins) account for 50 pct. or more of the dry weight of most living systems. A detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting projects have terminated at the crystal growth stage. In principle, there are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor is the elimination of density driven convective flow. Other factors that can be controlled in the absence of gravity is the sedimentation of growing crystals in a gravitational field, and the potential advantage of doing containerless crystal growth. As a result of these theories and facts, one can readily understand why the microgravity environment of an Earth orbiting vehicle seems to offer unique opportunities for the protein crystallographer. This perception has led to the establishment of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project. The results of experiments already performed during STS missions have in many cases resulted in large protein crystals which are structurally correct. Thus, the near term objective of the PCG/ME project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

Determination of the three-dimensional structure of proteins by crystallization and X-ray diffraction is becoming a valuable tool for revealing structure/function relationships that are of major importance in understanding how these macromolecules operate in biological systems. In recent years pharmaceutical, biotechnological, and chemical industries have become interested in crystallographic studies of proteins because of their promise in protein engineering, drug design, and other applications to biological systems. However, crystallization of most proteins and other biological macromolecules is a difficult process, and often the crystals obtained on the earth are too small for diffraction studies. Moreover, the crystals are poorly ordered and diffract to lower resolutions than those observed from most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of protein crystal growth experiments were performed on the U.S. Shuttle flights STS-26 in September 1988, STS-29 in March 1989, STS-32 in January 1990, and STS-31 in March 1990. For those proteins that produced crystals of adequate size, three-dimensional intensity data sets were collected with electronic area detector systems. Comparisons of the microgravity-grown crystals with the best earth-grown crystals obtained in numerous experiments demonstrate that when crystallization conditions are optimized, the microgravity-grown crystals of some proteins are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions. Analysis of the three-dimensional data sets by relative-Wilson plots indicates that the space-grown crystals are more highly ordered at the molecular level than their earth-grown counterparts.

Competitive inhibitors ofthesalvage path- wayenzymepurine-nucleoside phosphorylase (purine- nucleoside:orthophosphatese, EC 2.4.2.1) havebeen designed byusing thethree-dimensional strucre of theenzyme asdetermined byx-ray crystallography. The process wasanIterative onethat utilized interactive omputer graphics, MonteCarlo-based orational s ing,en- ergyminimization, andx-ray crystallography. Theproposed compounds weresynthesized andtested byaninvitro assay. Amongthecompounds dsigdandsynthesized arethemost potent competitive inhibitors ofpurine nucleoside phosphory- lase thusfarreported. Theconcept ofdrugdesign based oncrystallographic and modeling methods hasreceived muchattention, yetfewsolid examples haveappeared intheliterature. Twonotable ex- ceptions arethereports byErickson etal.(1) onthedesign ofhumanimmunodeficiency virus protease inhibitors and Appelt etal.(29) onthedesign ofthymidylate synthase inhibitors. Advances incrystallography, computer graphics, andrelated fields haveresulted inadramatic increase inthe number ofmacromolecular structure determinations while advances incomputer hardware andcomputational methods haveallowed thecomputational chemist toaddress more complex problems withhigher accuracy (2,3).Clearly, structural information combined withgraphical methods for depicting theaccessible volume, electrostatic potential, and active-site hydrophobicity aidsdrugdesign. Further en- hancement inthequality ofthe"designed compounds" is expected frommethods thatcanaccurately evaluate the target molecule intermsofbinding conformation, binding affinity, andbinding-induced changes inprotein conforma- tion. Wehavedeveloped MonteCarlo-based conformational search methods that, incombination withenergy minimiza- tion, accurately predict thecrystallographically observed ligand binding conformations forenzyme-inhibitor com- plexes. We nowreport theuseofcrystallographic and modeling methods forthedesign ofcompetitive inhibitors of theenzyme purine nucleoside phosphorylase (PNP; purine nucleoside:orthophosphate ribosyltransferase,

This invention relates generally to control systems for controlling crystal growth, and more particularly to such a system which uses a beam of light refracted by the fluid in which crystals are growing to detect concentration of solutes in the liquid. In a hanging drop apparatus, a laser beam is directed onto drop which refracts the laser light into primary and secondary bows, respectively, which in turn fall upon linear diode detector arrays. As concentration of solutes in drop increases due to solvent removal, these bows move farther apart on the arrays, with the relative separation being detected by arrays and used by a computer to adjust solvent vapor transport from the drop. A forward scattering detector is used to detect crystal nucleation in drop, and a humidity detector is used, in one embodiment, to detect relative humidity in the enclosure wherein drop is suspended. The novelty of this invention lies in utilizing angular variance of light refracted from drop to infer, by a computer algorithm, concentration of solutes therein. Additional novelty is believed to lie in using a forward scattering detector to detect nucleating crystallites in drop.