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.     

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.     

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.     

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.     

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.     

Macromolecular crystal growth experiments on International Microgravity Laboratory--1.

Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory--1 (IML-1), flown January 22-29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20-22 degrees C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes.     

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.     

The effects of serum and human albumin on calcium hydroxyapatite crystal growth.

The effects of potential serum inhibitors upon the growth of calcium hydroxyapatite (HAP) crystals were studied in vivo using a pH-stat system. Whole serum caused a marked decrease in crystal growth in a dose-dependent manner. At a protein concentration of 13 micrograms/ml, whole serum reduced the initial rate of crystal growth from 84 mumol of KOH/h to 48 mumol of KOH/h. Serum components were separated by ultrafiltration (10,000 Da cut-off). The high-molecular-mass fraction containing serum proteins gave an initial rate of crystal growth of 48 mumol of KOH/h compared with 64 mumol of KOH/h given by the low-molecular-mass components. Thus, two-thirds of the inhibitory activity was associated with proteins and other serum macromolecules, whilst the remainder of the activity was associated with the low-molecular-mass components. Albumin-depleted serum showed an initial rate of crystal growth of 59 mumol of KOH/h, whilst albumin purified by affinity chromatography gave an initial rate of crystal growth of 56 mumol of KOH/h at the same protein concentration. Albumin, therefore, not only accounts for half of the protein concentration in serum, but also contributes half of the inhibitory activity of the high-molecular-mass fraction. Heat denaturation of albumin dramatically enhanced the inhibition of HAP seeded growth with the initial rate of crystal growth falling to 27 mumol of KOH/h after treatment compared with 62 mumol of KOH/h before denaturation. Isoelectric focusing indicated that the tertiary and secondary structure, and hence the distribution of surface charge of albumin, are altered by heat denaturation. Gels showed a mixture of species with isoelectric points ranging from 6.0 to 5.0 compared with the native protein value of 4.7. These data suggest that adsorption of serum proteins to the growing HAP crystals is one mechanism of growth inhibition. It is also clear that the most abundant serum protein, albumin, is an important mediator of this process.     

固氮酶CrFe蛋白和MnFe蛋白的空间晶体生长

从分别生长于含Mn和Cr培养基中的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3分离纯化出MnFo和CrFe蛋白.为适应包括固氮酶在内的氧敏感蛋白的空间晶体生长的要求,应用简易而适用的厌氧加样装置代替固氮酶实验室所用的笨重厌氧箱(dry box),在地面进行厌氧加样.在充满氮气的简便有机玻璃箱内厌氧加样的所有样品中,分别用液/液扩散法和汽相扩散的坐滴法都可在一周内使MnFe和CrFe蛋白在宇宙飞船上从溶液中结晶出来.在所用的数种蛋白沉淀剂中,飞船上形成的所有晶体都为单晶,而地面上在多数沉淀剂中部生成大量孪晶.在相同沉淀剂中用液/液扩散法,飞船上生成CrFe蛋白的最大晶体比地面生成的最大晶体大1倍.而在相同沉淀剂中用汽相扩散的坐滴法,飞船上生成的MnFe蛋白最大晶体却没有地面生成的最大晶体大.这种差异也许是由不同结晶方法而不是不同蛋白所引起的.     

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

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A lipid's eye view of membrane protein crystallization in mesophases

Lipidic mesophases have been used to produce diffraction-quality crystals of several membrane proteins. The mechanism by which the method works is a mystery. The thrust is to continue to use it whilst deciphering the underlying mechanism and solving the second 'phase problem' in membrane protein crystallography. The method, which probably shares similarities with crystal growth in microgravity, is examined here from a lipid and a phase science perspective.     

Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium-binding proteins

Mineralization of bone matrix may be influenced by the presence of specific, noncollagenous bone proteins. The quantitative influence of two bone-specific proteins--bone gamma-carboxyglutamic acid (Gla) protein and osteonectin--and other proteins that decreased the rate of crystal growth was measured by adding seed crystals of hydroxyapatite to a solution of CaCl2 and KH2PO4, pH 7.4 at 37 degrees C. The molar concentrations of proteins needed to inhibit the rate of crystal growth by 50% were as follows: osteonectin, 0.15 microM; bone Gla protein, 0.8 microM; prothrombin, 0.9 microM; prothrombin fragment 1, 1.0 microM; soybean trypsin inhibitor, 3 microM; prethrombin 1, 9 microM; cytochrome c, 30 microM. Calmodulin and parvalbumin were found to be less active than prothrombin fragment 1 and had no activity in the micromolar range. The combination of two inhibitors resulted in a mixture with an inhibitory activity that was the sum of the two inhibitors. Decarboxylation of bone Gla protein significantly reduced its inhibitory activity. These results indicate that the inhibitory activity of a protein does not correlate with Ca2+-binding affinity under these conditions, that the mixture of inhibitors has an additive effect, and that gamma-carboxyglutamic acid residues enhance the ability of a protein to inhibit hydroxyapatite-seeded crystal growth.     

Crystallization of yeast iso-2-cytochrome c using a novel hair seeding technique

A hair seeding technique has been developed to obtain diffraction quality crystals of yeast (Saccharomyces cerevisiae) iso-2-cytochrome c, a model for studies of protein folding and biological electron transfer reactions. Deep red crystals of this protein were obtained from 88 to 92% saturated solutions of ammonium sulfate containing 20 mg protein/ml, 0.1 M-sodium phoshate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to phosphate, 0.3 M-sodium chloride, 0.04 M-dithiothreitol and adjusted to pH 6.0. Rapid crystal growth was observed, but only along the path of the seeding hair stroke. The space group is P4(3)2(1)2 (or P4(1)2(1)2) with a = b = 36.4 A, c = 137.8 A (1 A = 0.1 nm) and Z = 8. Crystals are stable in the X-ray beam for more than 10 days and diffract to at least 2.5 A resolution. The same hair seeding methodology has proven useful in obtaining crystals of specifically designed mutant iso-2 proteins and in other protein systems where consistent crystal growth had previously proven difficult to attain.     

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.     

Crucial steps in the structure determination of the Na+/H+ antiporter NhaA in its native conformation

Sodium proton antiporters are ubiquitous membrane proteins. Their importance for cell viability is the result of their role in homeostasis of intracellular pH, cellular Na+ content and cell volume. Recently, the first structure of this family of secondary transporters, namely of NhaA from Escherichia coli, revealed a novel fold and elucidated the molecular basis for the mechanism of transport and its regulation by pH. Here, we describe the key steps for the structure determination of NhaA, an iterative process of improving protein quality as well as crystallization conditions. Protein quality was optimized by shortening the purification to a single step and by changing the expression host. The major steps for crystal improvement were the exchange of the detergent during protein purification from the beta- to the alpha-anomer of DDM, the addition of OG to the crystallization set ups, and the growth of the crystals under conditions suitable for cryo-temperatures. Unexpectedly, the dimeric association of the transporter in the 3D crystal lattice is non-physiological. A comparison of the X-ray structure with the electron density map from cryo-electron microscopy of 2D crystals demonstrates that the NhaA helix packing in the 3D crystal is identical with the one in the lipid environment. Thus, the antiporter is in a native conformation in the 3D crystals.     

Protein crystal growth. Growth kinetics for tetragonal lysozyme crystals

A method for immobilizing protein crystals has been devised for determining face growth rates, and used to investigate the growth kinetics of hen egg white lysozyme crystals. Growth rates were determined at 22 degrees C in 0.1 M sodium acetate, 5% NaCl, pH 4.0, on the visually identified (110) face of tetragonal lysozyme crystals. Protein concentrations ranged from 13 to 57 mg/ml (saturation concentration = 1.7 mg/ml). Growth rate data were fit to the equation R = kappa sigma ri, where R = rate in cm/s; kappa = constant; sigma i = solute growth interface supersaturation; and r = rate dependence upon super-saturation, with the result that kappa = 0.146 X 10(-8) cm/s and r = 2.0. A model of the growth process was developed and the experimental data were used to determine the relative roles of transport and interfacial kinetics in the growth of this crystal. Values for the width of the boundary layer delta, the interfacial concentration Ci, and growth rate R were determined. The model may be used to extrapolate to other growth conditions. The relative role of transport and interfacial kinetics can be expressed by the coefficient gamma = (CB - Ci)/(CB - Cs), when CB is the bulk concentration and Cs the saturation. Values for gamma were found to range from much less than 0.1 for submicron-size crystals to approximately 0.15 for cm sizes. The results indicate that attachment or surface effects are rate-limiting in lysozyme crystal growth in Earth's gravity because solutal convection always provides more transport of solute than can be accommodated by the interface. In order to grow such crystals under transport limiting conditions, it would be necessary to suppress this solutal convection.     

Surface heat shock protein 90 serves as a potential receptor for calcium oxalate crystal on apical membrane of renal tubular epithelial cells

Adhesion of calcium oxalate monohydrate (COM) crystals on renal tubular epithelial cells is a crucial step in kidney stone formation. Finding potential crystal receptors on the apical membrane of the cells may lead to a novel approach to prevent kidney stone disease. Our previous study identified a large number of crystal-binding proteins on the apical membrane of MDCK cells. However, their functional role as potential crystal receptors had not been validated. The present study aimed to address the potential role of heat shock protein 90 (HSP90) as a COM crystal receptor. The apical membrane was isolated from polarized MDCK cells by the peeling method and recovered proteins were incubated with COM crystals. Western blot analysis confirmed the presence of HSP90 in the apical membrane and the crystal-bound fraction. Immunofluorescence staining without permeabilization and laser-scanning confocal microscopy confirmed the surface HSP90 expression on the apical membrane of the intact cells. Crystal adhesion assay showed that blocking surface HSP90 by specific anti-HSP90 antibody and knockdown of HSP90 by small interfering RNA (siRNA) dramatically reduced crystal binding on the apical surface of MDCK cells (by approximately 1/2 and 2/3, respectively). Additionally, crystal internalization assay revealed the presence of HSP90 on the membrane of endocytic vesicle containing the internalized COM crystal. Moreover, pretreatment of MDCK cells with anti-HSP90 antibody significantly reduced crystal internalization (by approximately 1/3). Taken together, our data indicate that HSP90 serves as a potential receptor for COM crystals on the apical membrane of renal tubular epithelial cells and is involved in endocytosis/internalization of the crystals into the cells.     

Altered proteins in MDCK renal tubular cells in response to calcium oxalate dihydrate crystal adhesion: a proteomics approach

The interaction between crystals and renal tubular cells has been proposed to be a crucial event that elicits subsequent cellular responses, leading to kidney stone formation. Nevertheless, the molecular mechanisms of these cellular responses remain poorly understood. We performed a gel-based differential proteomics study to examine cellular responses (as determined by altered protein expression) in Madin-Darby canine kidney (MDCK) cells, which were derived from dog kidney and exhibited distal renal tubule phenotype, during calcium oxalate dihydrate (COD) crystal adhesion. MDCK cells were grown in a medium without or with COD crystals (100 microg/ml) for 48 h. Crystal adhesion was illustrated by phase-contrast and scanning electron microscopy. Flow cytometry using annexin V/propidium iodide double staining showed that the percentage of cell death did not significantly differ between cells with and without COD crystal adhesion. Cellular proteins were then extracted, resolved with two-dimensional gel electrophoresis (2-DE), and visualized by SYPRO Ruby staining ( n = 5 gels per group). Quantitative intensity analysis revealed 11 significantly altered proteins, 10 of which were successfully identified by quadrupole time-of-flight peptide mass fingerprinting (MS) and/or tandem MS (MS/MS), including metabolic enzymes, cellular structural protein, calcium-binding protein, adhesion molecule, protein involved in RNA metabolism, and chaperone. An increase in annexin II was confirmed by 2-D Western blot analysis. These data may lead to better understanding of the cellular responses in distal renal tubular cells during COD crystal adhesion.     

Fusion protein approach to improve the crystal quality of cytochrome bo(3) ubiquinol oxidase from Escherichia coli

Crystals of cytochrome bo(3) ubiquinol oxidase from E. coli diffract X-rays to 3.5 A and the structure determination is in progress. The limiting factor to the elucidation of the structural detail is the quality of the crystals; the diffraction spots from the crystals are diffused which leads to difficulties in processing the data beyond 4.0 A. Weak protein-protein contacts within the crystal lattice is assumed to be the cause of this problem. To improve these contacts, we have introduced protein Z to the C-terminal end of the subunit IV of cytochrome bo(3) and expressed both proteins as a single fusion. We have successfully obtained crystals of this fusion protein. The spot shape problem has clearly been solved in the crystals of the fusion protein although further optimization is necessary to obtain higher resolution. We also discuss the potential applications of this approach to the crystallization of membrane proteins in general.     

Calcium oxalate dihydrate crystal induced changes in glycoproteome of distal renal tubular epithelial cells

Calcium oxalate dihydrate (COD) crystals can adhere onto the apical surface of renal tubular epithelial cells. This process is associated with crystal growth and aggregation, resulting in kidney stone formation. Glycoproteins have been thought to play roles in response to crystal adhesion. However, components of the glycoproteome that are involved in this cellular response remain largely unknown. Our present study therefore aimed to identify altered glycoproteins upon COD crystal adhesion onto tubular epithelial cells representing distal nephron, the initiating site of kidney stone formation. Madin-Darby Canine Kidney (MDCK) cells were maintained in culture medium with or without COD crystals for 48 h (n = 5 flasks per group). Cellular proteins were extracted, resolved by 2-DE and visualized by SYPRO Ruby total protein stain, whereas glycoproteins were detected by Pro-Q Emerald glycoprotein dye. Spot matching and quantitative intensity analysis revealed 16 differentially expressed glycoprotein spots, whose corresponding total protein levels were not changed by COD crystal adhesion. These altered glycoproteins were successfully identified by Q-TOF MS and/or MS/MS analyses, and potential glycosylation sites were identified by the GlycoMod tool. For example, glycoforms of three proteasome subunits (which have a major role in regulating cell-cell dissociation) were up-regulated, whereas a glycoform of actin-related protein 3 (ARP3) (which plays an important role in cellular integrity) was down-regulated. These coordinated changes implicate that COD crystal adhesion induced cell dissociation and declined cellular integrity in the distal nephron. Our findings provide some novel insights into the pathogenic mechanisms of kidney stone disease at the molecular level, particularly cell-crystal interactions.