Protein crystal growth in microgravity.

The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for 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 11 different protein crystal growth experiments was performed on U.S. space shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on Earth.

Purification of octyl beta-D-glucopyranoside and re-estimation of its micellar size.

The commercial non-ionic detergent octyl beta-D-glucopyranoside is often contaminated by significant amounts of UV absorbing and/or ionic compounds that can associate with membrane proteins. Such impurities can be monitored by several techniques (i.e., spectrophotometry, size exclusion chromatography, and pH, conductivity, and surface tension measurements) and can be removed using mixed-bed ion exchange chromatography. High performance size exclusion chromatography, dynamic light scattering, and ultracentrifugation have been used to re-estimate the size of micelles of octyl beta-D-glucopyranoside since previously published data varied over a wide range. Aggregation numbers were 27 to 100 for micellar molecular weights 8000 to 29,000. Direct physical methods that do not perturbate the sample indicated a large size for the micelles (hydrodynamic radius 23 +/- 3 A; Mr 22,000 +/- 3000; aggregation number 75 +/- 10 for a 34 mM aqueous solution). In contrast the chromatographic micellar size appeared to be smaller (hydrodynamic radius 15 +/- 1 A; Mr 8000 +/- 1000; aggregation number 27). This underestimation may be the result of adsorption and/or alteration of the micelles.

Preliminary crystallographic studies on human complement pro-factor D.

The recombinant zymogen of the human complement protein factor D has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 as precipitant. Two crystal forms obtained at pH 5.4 belong to space group P2(1). The crystals grow to dimensions of 0.6 mm x 0.3 mm x 0.3 mm in three days, are stable in the X-ray beam, and diffract to 2.4 A.

Crystallization and preliminary X-ray investigation of factor D of human complement.

Human factor D, an essential enzyme of the alternative pathway of complement activation, has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 and NaCl as precipitants. The factor D crystals are triclinic and the space group is P1 with unit cell dimensions a = 40.8 A, b = 64.7 A, c = 40.3 A, alpha = 101.0 degrees, beta = 109.7 degrees, gamma = 74.3 degrees. The unit cell contains two molecules of factor D related by a non-crystallographic 2-fold axis. The crystals grow to dimensions of 0.8 mm x 0.5 mm x 0.2 mm within five days, are stable in the X-ray beam and diffract beyond 2.5 A.

Structure of human factor D. A complement system protein at 2.0 A resolution.

Factor D, an essential enzyme for the activation of the alternative pathway of the complement system, belongs to the serine protease superfamily. The crystal structure of the enzyme was solved by a combination of multiple isomorphous replacement and molecular replacement methods. The present model was refined to an R-factor of 18.8% using 23,681 observed reflections between 7.5 and 2.0 A resolution, with a root-mean-square deviation from standard bond lengths of 0.016 A. The two non-crystallographically related molecules in the triclinic unit cell have distinctive active site conformations. The protein has the general structural fold of a serine protease, but there are several unique amino acid substitutions resulting in significant alterations in the critical loops responsible for catalysis and substrate specificity in serine proteases. Factor D is the first complement serine protease whose three-dimensional structure has been determined.

Preliminary X-ray study of crystals of human C-reactive protein.

Two different crystal forms of human C-reactive protein have been grown from solutions of 2-methyl-2,4-pentanediol. Both crystal forms are tetragonal, the space group for form I is P4(1)22 (or P4(3)22), and that for form II is P4(2)22. The unit cell parameters for form I are a = b = 103.0(5) A, c = 308.5(7) A and for form II are a = b = 103.1(2) A, c = 312.7(6) A. The crystals of form II diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.

Rotation function studies of human C-reactive protein.

Rotation function studies of two tetragonal crystal forms of human C-reactive protein have confirmed the pentameric structure of the molecule. The two crystal forms have space groups P4122 (I) and P4222 (II) with closely similar unit cells and are often twinned together. Investigation of the crystallization conditions indicates that dissociation heterogeneity has been a major limiting factor in the reproducible growth of good single crystals. The orientation of the pentameric molecule is shown to be almost identical in both forms, about the axial direction omega = 57 degrees, phi = 45 degrees, i.e. 57 degrees away from c in the (110) plane.

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase.

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.

Crystal structure of adenosine kinase from Toxoplasma gondii at 1.8 A resolution.

Human infection with Toxoplasma gondii is an important cause of morbidity and mortality. Protozoan parasites such as T. gondii are incapable of de novo purine biosynthesis and must acquire purines from their host, so the purine salvage pathway offers a number of potential targets for antiparasitic chemotherapy. In T. gondii tachyzoites, adenosine is the predominantly salvaged purine nucleoside, and thus adenosine kinase is a key enzyme in the purine salvage pathway of this parasite. The structure of T. gondii adenosine kinase was solved using molecular replacement and refined by simulated annealing at 1.8 A resolution to an R-factor of 0.214. The overall structure and the active site geometry are similar to human adenosine kinase, although there are significant differences. The T. gondii adenosine kinase has several unique features compared to the human sequence, including a five-residue deletion in one of the four linking segments between the two domains, which is probably responsible for a major change in the orientation of the two domains with respect to each other. These structural differences suggest the possibility of developing specific inhibitors of the parasitic enzyme.

Large scale preparation of homogeneous bacteriorhodopsin.

Homogeneous bacteriorhodopsin was obtained preparatively (100 mg batches) from purple membrane of Halobacterium halobium cells. The homogeneity of the protein was considerably affected by variations in the growth conditions of the bacteria. Fully matured bacteriorhodopsin having a blocked N-terminus and a homogeneous C-terminus, was reproducibly obtained when cells were grown in a sufficiently aerated medium.

Bioactivity of peptide analogs of the neutrophil chemoattractant, N-acetyl-proline-glycine-proline.

PURPOSE: The release of N-acetyl-proline-glycine-proline (PGP), a chemoattractant resulting from direct alkaline hydrolysis of corneal proteins, is believed to be the initial trigger for neutrophil invasion into the alkali-injured cornea. The purpose of this study is twofold: (1) to compare the activity of N-acetyl-PGP with the bioactivities of other similar synthetic peptides in an effort to uncover information about this chemoattractant molecule, and (2) to test these peptide analogs as potential antagonists of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of human neutrophils to peptides. Bioactivity was expressed as the peptide concentration required to produce 50% neutrophil polarization (EC50). Antagonist activity was expressed as the peptide concentration required to produce 50% inhibition (ID50) of polarization activated by N-acetyl-PGP. RESULTS: Peptide bioactivities (EC50) were ranked as follows: APGPR (0.34 mM) > N-acetyl-PGP (0.5 mM) > N-(PGP)4-PGLG (3 mM) = t-Boc-PGP (3 mM) > N-acetyl-PG (3.4 mM) > N-methyl-PGP (15 mM) = PGP (15 mM) > peptides without detectable activity (t-Boc-PGP-OMe, N-acetyl-P, PG, PGG, GP, GG and gly-pro-hyp). Peptides with no detectable bioactivity were tested as potential antagonists of neutrophil polarization induced by N-acetyl-PGP. Gly-Pro-Hyp inhibited N-acetyl-PGP activation of polarization at 20 mM (ID50). No other synthetic peptide demonstrated a capacity for inhibition. CONCLUSIONS: The minimum requirement to elicit bioactivity was the presence of PGP alone or derivatives of PG in which the N-terminal proline is blocked. Using this approach, active and inactive mimetic peptides of N-acetyl-PGP were produced. The most active peptide, APGPR, was equal to or slightly greater than N-acetyl-PGP, suggesting that more potent analogs might be designed. Gly-pro-hyp was the only inactive peptide analog to inhibit the chemoattractant.

Protein crystal growth in microgravity.

Protein crystallography is a powerful method for determining the three-dimensional structures of biological macromolecules. Although new methods, such as two-dimensional NMR, have demonstrated promise for determining the structures of small proteins and nucleic acids, the complete atomic arrangements within large proteins can only be determined at present using crystallographic techniques. Such crystallographic studies have been of major importance for establishing structure/function relationships that are fundamental to understanding how enzymes, nucleic acids, and other macromolecules function in biological systems. More recently, crystallographic studies of proteins have become of considerable practical interest within the pharmaceutical and biotechnology industries, as promising tools in drug design and in protein engineering.

Large-scale preparation of the delta10 form of staphylokinase by in vitro processing of recombinant staphylokinase with purified human plasminogen.

The authors have developed a rapid and convenient method for purification of a low molecular weight form (delta 10) of the bacterial plasminogen activator, staphylokinase. Recombinant staphylokinase is expressed in Escherichia coli, with an amino terminal extension that facilitated purification by immobilized metal-affinity chromatography. Purified staphylokinase is treated with human plasminogen, and the resulting truncated form is purified using a combination of immobilized metal affinity chromatography and hydrophobic interaction chromatography. Purified protein is characterized by amino terminal sequencing and in vitro plasminogen activation assay.

Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32.

NASA: The first microgravity protein crystal growth experiments were performed on Spacelab I by Littke and John. These experiments indicated that the space grown crystals, which were obtained using a liquid-liquid diffusion system, were larger than crystals obtained by the same experimental system on earth. Subsequent experiments were performed by other investigators on a series of space shuttle missions from 1985 through 1990. The results from two of these shuttle flights (STS-26 and STS-29) have been described previously. The results from these missions indicated that the microgravity grown crystals for a number of different proteins were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth. This paper presents the results obtained from shuttle flight STS-32 (flown in January, 1990) and preliminary results from the most recent shuttle flight, STS-31 (flown in April, 1990).^ieng

Protein crystal growth and the International Space Station.

Protein structural information plays a key role in understanding biological structure-function relationships and in the development of new pharmaceuticals for both chronic and infectious diseases. The Center for Macromolecular Crystallography (CMC) has devoted considerable effort studying the fundamental processes involved in macromolecular crystal growth both in a 1-g and microgravity environment. Results from experiments performed on more than 35 U.S. space shuttle flights have clearly indicated that microgravity can provide a beneficial environment for macromolecular crystal growth. This research has led to the development of a new generation of pharmaceuticals that are currently in preclinical or clinical trials for diseases such as cutaneous T-cell lymphoma, psoriasis, rheumatoid arthritis, AIDS, influenza, stroke and other cardiovascular complications. The International Space Station (ISS) provides an opportunity to have complete crystallographic capability on orbit, which was previously not possible with the space shuttle orbiter. As envisioned, the x-ray Crystallography Facility (XCF) will be a complete facility for growing protein crystals; selecting, harvesting, and mounting sample crystals for x-ray diffraction; cryo-freezing mounted crystals if necessary; performing x-ray diffraction studies; and downlinking the data for use by crystallographers on the ground. Other advantages of such a facility include crystal characterization so that iterations in the crystal growth conditions can be made, thereby optimizing the final crystals produced in a three month interval on the ISS.

Mercury induced modifications in the stereochemistry of the active site through Cys-73 in a serine protease--crystal structure of the complex of a partially modified proteinase K with mercury at 1.8 A resolution.

Proteinese K (PK) isolated from Tritirachium album Limber was crystallized with HgCl2 in excess, under microgravity conditions. The intensity data were collected at 4 degrees C to 1.8 A resolution and the final R-factor after refinement for all the reflections was 0.164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 A and 2.58 A respectively from Cys-73 Sgamma. The Cys-73 in the enzyme structure is located close to the active site residue, His-69. This region is completely buried and is not accessible to the solvent. It is rather tightly packed. Therefore, the binding of mercury distorts the stereochemistry of the neighbouring residues including those belonging to the catalytic triad. As a result of this, the Ogamma of Ser-224 is displaced by 0.6 A which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 A between Ser-224 Ogamma and His-69 Nepsilon2.

Crystallization of the EGF receptor ectodomain on US space mission STS-47.

Although biochemists working in the field of biological signal transduction have characterized cell surface receptors for numerous growth factors within the past ten years, none of the three-dimensional structures could be obtained for these important proteins which represent major components of the cells' growth control system. Now, the extracellular ligand binding domain of the EGF receptor was crystallized in the presence of EGF under microgravity on US Shuttle mission STS-47. In 8 out of 9 experiments prepared under different conditions crystal growth was observed. One of these space-grown crystals showed higher diffraction quality than all crystals previously obtained in the laboratory. It allowed, for the first time, evaluation of the real space group by partial data collection.

Protein crystal growth in microgravity-temperature induced large scale crystallization of insulin.

One of the major stumbling blocks that prevents rapid structure determination using x-ray crystallography is macromolecular crystal growth. There are many examples where crystallization takes longer than structure determination. In some cases, it is impossible to grow useful crystals on earth. Recent experiments conducted in conjunction with NASA on various Space Shuttle missions have demonstrated that protein crystals often grow larger and display better internal molecular order than their earth-grown counterparts. This paper reports results from three Shuttle flights using the Protein Crystallization Facility (PCF). The PCF hardware produced large, high-quality insulin crystals by using a temperature change as the sole means to affect protein solubility and thus, crystallization. The facility consists of cylinders/containers with volumes of 500, 200, 100, and 50 ml. Data from the three Shuttle flights demonstrated that larger, higher resolution crystals (as evidenced by x-ray diffraction data) were obtained from the microgravity experiments when compared to earth-grown crystals.