Equilibration of precipitants in a counter-diffusion apparatus for protein crystallization.

A cost-effective capillary dialysis apparatus (Toledo Capillary Box, TCB) developed for biomacromolecule crystal growth in microgravity and unit gravity environments can provide slow equilibration between the precipitant reservoir and capillary solutions, nurturing growth of neutron-diffraction-quality crystals. Under microgravity conditions, mass transfer of precipitants and biomacro-mol-ecules occurs under diffusion-controlled conditions, promoting slow growth and suppressing defect formation. The equilibration of common precipitants (polyethyl-ene glycol and salts such as ammonium sulfate) between capillary and reservoir solutions was measured for capillaries oriented horizontally or vertically with respect to the gravitational field at unit gravity. Precipitants equilibrated less rapidly in the vertical orientation when capillary solution densities were lower than those of the reservoir solutions. A plug filled with agarose gel was introduced in the TCB apparatus for salt precipitants since salts often exhibit relatively high free diffusion. Equilibration of the capillaries with reservoir solutions was significantly delayed for many of the salt precipitants tested. Analytical and semi-analytical models allow the prediction of precipitant equilibration of capillary and reservoir solutions under diffusion-controlled transport and show good agreement with experimental results.

Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction.

Biologically active vitamin B6-derivative pyridoxal 5'-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm3). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15.

Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass.

Critical cellulase and hemicellulase activities are identified for hydrolysis of ionic liquid (IL) pretreated poplar and switchgrass; hemicellulase rich substrates with largely amorphous cellulose. Enzymes from Aspergillus nidulans were expressed and purified: an endoglucanase (EG) a cellobiohydrolase (CBH), an endoxylanase (EX) and an acetylxylan esterase (AXE). ß-Xylosidase (ßX) from Selenomonas ruminantium and a commercial ß-glucosidase (ßG) from Novozyme 188 were admixed with the A. nidulans enzymes. Statistical analysis indicates that ßG and ßX activities are significant for both glucose and xylose yields for the two substrates. EG is a significant factor for glucan hydrolysis while EX is significant for xylan hydrolysis of the substrates. The CBH, which has activity on crystalline cellulose and negligible activity on amorphous cellulose, was not a significant factor in glucan hydrolysis. EX is significant in glucan hydrolysis for poplar. The addition of AXE significantly improves xylan hydrolysis for poplar but not switchgrass.

Ionic-liquid induced changes in cellulose structure associated with enhanced biomass hydrolysis.

The effects of varying ionic liquid pretreatment parameters on various sources of lignocellulosic biomass have been studied using X-ray powder diffraction, X-ray fiber diffraction, and compositional analysis. Comparative enzymatic hydrolysis and sugar analysis were used to relate the observed changes in cellulose structure to biomass digestibility. In this study, the factor most clearly associated with enhanced biomass hydrolysis is the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase.

Reversible swelling of the cell wall of poplar biomass by ionic liquid at room temperature.

Time-resolved autofluorescence, Raman microspectroscopy, and scanning microprobe X-ray diffraction were combined in order to characterize lignocellulosic biomass from poplar trees and how it changes during treatment with the ionic liquid 1-n-ethyl-3-methylimidazolium acetate (EMIMAC) at room temperature. The EMIMAC penetrates the cell wall from the lumen, swelling the cell wall by about a factor of two towards the empty lumen. However, the middle lamella remains unchanged, preventing the cell wall from swelling outwards. During this swelling, most of the cellulose microfibrils are solubilized but chain migration is restricted and a small percentage of microfibrils persist. When the EMIMAC is expelled, the cellulose recrystallizes as microfibrils of cellulose I. There is little change in the relative chemical composition of the cell wall after treatment. The action of EMIMAC on the poplar cell wall at room temperature would therefore appear to be a reversible swelling and a reversible decrystallization of the cell wall.

Saccharification of ionic liquid pretreated biomass with commercial enzyme mixtures.

The performance of Spezyme CP, a commercial cellulase system, and Primafast Luna CL, a textile bio-finishing enzyme, on hydrolysis of substrates following ionic liquid pretreatment was evaluated. Ionic liquid pretreatment of lignocellulosic biomass produces amorphous cellulose with little residual crystallinity and enhances its saccharification. The high crystallinity of native cellulose poses an impediment to enzyme hydrolysis of the cellulosic portion of biomass. Target substrates included poplar, switchgrass and Avicel, a highly crystalline cellulose substrate. Spezyme was found to hydrolyze glucan and xylan components completely in 24h with modest enzyme loadings. Primafast was found to principally hydrolyze glucan components of biomass. Xylanases added to the Primafast enzyme mixture appear to act synergistically to improve glucan hydrolysis with the poplar substrate.

Beneficial effect of solubility enhancers on protein crystal nucleation and growth.

Crystallizing solutions of proteins often contain various nonelectrolyte additives that arise from the purification process of proteins or from the reagents employed in the screening kits. Currently, limited knowledge exists about the influence of these additives on the mechanisms underlying the crystallization process, in particular on the nucleation stage of crystals. To address this need, we studied crystallization of two proteins, D-xylose isomerase and chicken egg-white lysozyme, in small batches and in the presence of two solubility-enhancing additives, acetonitrile and glycerol. We have also measured the nucleation rates of crystals of these proteins in the presence and in the absence of acetonitrile using the method of initial rates. With the addition of the solubility enhancers, both proteins exhibited an increase in crystal nucleation at any given supersaturation. Solubility enhancing additives appear to lower the energy barrier to nucleation by influencing the strength of attraction between the protein molecules. We have characterized the quality of D-xylose isomerase crystals by determining the crystal mosaicity, which showed considerable improvement for crystals grown in the presence of additives. When compared to the crystals of chicken egg-white lysozyme, D-xylose isomerase crystals required higher supersaturations to nucleate. We attribute this result to the large size of the D-xylose isomerase molecule, which influences the energy barrier to nucleation by increasing the surface area of the critical nucleus. Contrary to the common expectation that reagents that solubilize the protein may hinder the crystallization process, our results suggest that solubility enhancers, in fact, can have a beneficial effect on the nucleation and growth of crystals. These findings are of importance in formulating successful strategies toward crystallizing new proteins.

Determination of ethanol in ionic liquids using headspace solid-phase microextraction-gas chromatography.

The application of ionic liquids (ILs) as nonderivatizing solvents for the pretreatment and regeneration of cellulose is a growing area of research. Here we report the development of a rapid and simple method for the determination of residual ethanol content in two hydrophilic ILs, 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium acetate. The method utilizes headspace solid-phase microextraction coupled with gas chromatography at elevated extraction temperatures, resulting in rapid equilibration times. The effect of IL water content on the ethanol extraction efficiency is presented. Recovery experiments carried out in real samples gave recoveries ranging from 96.8 to 98.2%.

Optimization of buffer solutions for protein crystallization.

Increasing the solubility of protein stock solutions to above that in a standard chromatography buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl) led to an increase in the number of crystallization conditions for ten globular proteins subjected to two crystal screens: the Index and Precipitant/Precipitant-Additive (P/PA) Screens. Solubility enhancement of protein stock solutions was achieved through screening and selection of buffer components to formulate an optimal buffer. Relative improvements in solubility were estimated through protection against the precipitation of protein by polyethylene glycol 8000. Proteins with limited solubility improvement in optimal buffer showed an enhancement in solubility on addition of glycerol. Maximum solubility was then determined by the concentration of optimized solutions until precipitate formed. The supernatant concentration then provided an estimate of the upper limit of protein solubility. This 'solubility' estimate is used to specify the initial concentration of the protein used in the screening experiments and is an important step in successful crystallization. Buffer optimization and establishment of initial protein concentration for crystal screening based on solubility estimates provides a methodology for improved crystal screening results.

Cryogenic (<20 K) helium cooling mitigates radiation damage to protein crystals.

In experiments conducted at the Bio-CARS beamline 14-BM-C (APS, Argonne National Laboratory, USA), Streptomyces rubiginosus D-xylose isomerase (EC 5.3.1.5) crystals were used to test the effect of cryogen temperature on radiation damage. Crystals cooled using a helium cryostat at an 8 K set temperature consistently showed less decay in the signal-to-noise ratio, I/sigma(I), and in average intensity, I, compared with those cooled with a nitrogen cryostat set to 100 K. Multiple crystals grown using ammonium sulfate as precipitant were used at each cryostat set temperature and comparisons were made for crystals of similar size and diffraction resolution. Maximum resolution for the crystals was 1.1-1.3 A, with He at <20 K extending the lifetime of the high-resolution data by >25% compared with crystals cooled with N(2) at 100 K.

Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment.

Efficient hydrolysis of cellulose-to-glucose is critically important in producing fuels and chemicals from renewable feedstocks. Cellulose hydrolysis in aqueous media suffers from slow reaction rates because cellulose is a water-insoluble crystalline biopolymer. The high-crystallinity of cellulose fibrils renders the internal surface of cellulose inaccessible to the hydrolyzing enzymes (cellulases) as well as water. Pretreatment methods, which increase the surface area accessible to water and cellulases are vital to improving the hydrolysis kinetics and conversion of cellulose to glucose. In a novel technique, the microcrystalline cellulose was first subjected to an ionic liquid (IL) treatment and then recovered as essentially amorphous or as a mixture of amorphous and partially crystalline cellulose by rapidly quenching the solution with an antisolvent. Because of their extremely low-volatility, ILs are expected to have minimal environmental impact. Two different ILs, 1-n-butyl-3-methylimidazolium chloride (BMIMC1) and 1-allyl-3-methylimidazolium chloride (AMIMC1) were investigated. Hydrolysis kinetics of the IL-treated cellulose is significantly enhanced. With appropriate selection of IL treatment conditions and enzymes, the initial hydrolysis rates for IL-treated cellulose were up to 90 times greater than those of untreated cellulose. We infer that this drastic improvement in the "overall hydrolysis rates" with IL-treated cellulose is mainly because of a significant enhancement in the kinetics of the "primary hydrolysis step" (conversion of solid cellulose to soluble oligomers), which is the rate-limiting step for untreated cellulose. Thus, with IL-treated cellulose, primary hydrolysis rates increase and become comparable with the rates of inherently faster "secondary hydrolysis" (conversion of soluble oligomers to glucose).

Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step.

Hydrolysis of cellulose to glucose in aqueous media catalyzed by the cellulase enzyme system suffers from slow reaction rates due in large part to the highly crystalline structure of cellulose and inaccessibility of enzyme adsorption sites. In this study, an attempt was made to disrupt the cellulose structure using the ionic liquid (IL), 1-n-butyl-3-methylimidazolium chloride, in a cellulose regeneration strategy which accelerated the subsequent hydrolysis reaction. ILs are a new class of non-volatile solvents that exhibit unique solvating properties. They can be tuned to dissolve a wide variety of compounds including cellulose. Because of their extremely low volatility, ILs are expected to have minimal environmental impact on air quality compared to most other volatile solvent systems. The initial enzymatic hydrolysis rates were approximately 50-fold higher for regenerated cellulose as compared to untreated cellulose (Avicel PH-101) as measured by a soluble reducing sugar assay.

Assessment of a preliminary solubility screen to improve crystallization trials: uncoupling crystal condition searches.

The utility of a preliminary solubility screen has been assessed on ten test proteins. It is proposed that maximizing the protein solubility prior to crystal setups is likely to improve crystal growth. In crystallization setups, drops of a protein solution are mixed with various crystallization solutions which are then allowed to equilibrate. The protein solutions usually contain a salt and buffer which are present as a constant in all crystal screens. The propensity for crystallization, driven by three components of sparse-matrix screens, the buffers, salts and precipitating agents, could potentially be masked by the components of the protein solution. Ten test proteins were dissolved in a standard buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.5) and in customized optimal buffers determined to maximize solubility. The proteins were then subjected to the Index (Hampton Research) 96-well sparse-matrix crystal screen and to a precipitant/precipitant-additive screen described here. Five of the ten proteins studied showed twofold to fourfold increases in the saturation level from standard to optimal buffer, two showed slight improvement and three showed a slight decrease. Microcrystals were obtained for all proteins and optimal buffer increased the appearance of crystals for eight of the ten proteins.

Protein crystal nucleation: is the pair interaction potential the primary determinant of kinetics?

Fundamental understanding of protein crystal nucleation facilitates crystallization of biological macromolecules for structure determination and control of crystal size distribution. In the studies presented here, nucleation kinetics of hen egg-white lysozyme crystals were measured at solution conditions that exhibited equal solubility by adjusting pH, temperature, or sodium chloride concentration. It was observed that solution conditions that lead to equal solubility resulted in equal nucleation rates and hence kinetic parameters. Since the solubility of globular proteins correlates with the osmotic second virial coefficient, B(22), an integral measure of the protein pair interaction potential, this observation indicates that the protein pair interaction plays a key role in determining nucleation kinetic parameters.

New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium.

Cryocrystallography is used today for almost all X-ray diffraction data collection at synchrotron beam lines, with rotating-anode generators, and micro X-ray sources. Despite the widespread use of flash-cooling to place macromolecular crystals in the cryogenic state, its use can ruin crystals, trips to the synchrotron, and sometimes even an entire project. Annealing of macromolecular crystals takes little time, requires no specialized equipment, and can save crystallographic projects that might otherwise end in failure. Annealing should be tried whenever initial flash-cooling causes an unacceptable increase in mosaicity, results in ice rings, fails to provide adequate diffraction quality, or causes a crystal to be positioned awkwardly. Overall, annealing improves the quality of data and overall success rate at synchrotron beam lines. Its use should be considered whenever problems arise with a flash-cooled crystal. Helium is a more efficient cryogen than nitrogen and will deliver lower temperatures. Experiments suggest that when crystals are cooled with He rather than N2, crystals maintain order and high-resolution data are less affected by increased radiation load. Individually or in combination, these two techniques can enhance the success of crystallographic data collection, and their use should be considered essential for high-throughput programs.

Experiments testing the abatement of radiation damage in D-xylose isomerase crystals with cryogenic helium.

Helium is a more efficient cryogen than nitrogen, and for macromolecular data collection at high-flux beamlines will deliver lower temperatures. An open-flow helium cryostat developed at the University of Toledo (the Pinkerton Device) has been used for macromolecular data collection. This device differs from standard commercial He cryostats by having a much narrower aperture providing a high velocity stream of He around the crystal that maximizes convective and conductive heat exchange between the crystal and the cryogen. This paper details a series of experiments conducted at the IMCA-CAT 17ID beamline using one crystal for each experimental condition to examine whether helium at 16 K provided better radiation-damage abatement compared with nitrogen at 100 K. These studies used matched high-quality crystals (0.94 A diffraction resolution) of D-xylose isomerase derived from the commercial material Gensweet SGI. Comparisons show that helium indeed abates the indicators of radiation damage, in this case resulting in longer crystal diffractive lifetimes. The overall trend suggests that crystals maintain order and that high-resolution data are less affected by increased radiation load when crystals are cooled with He rather than N(2). This is probably the result of a lower effective temperature at the crystal with concomitant reduction in free-radical diffusion. Other features, such as an apparent phase transition in macromolecular crystals at lower temperatures, require investigation to broaden the utility of He use.