Efficient protein crystallization

High-throughput molecular biology and crystallography advances have placed an increasing demand on crystallization, the one remaining bottleneck in macromolecular crystallography. This paper describes three experimental approaches, an incomplete factorial crystallization screen, a high-throughput nanoliter crystallization system, and the use of a neural net to predict crystallization conditions via a small sample (approximately 0.1%) of screening results. The use of these technologies has the potential to reduce time and sample requirements. Initial experimental results indicate that the incomplete factorial design detects initial crystallization conditions not previously discovered using commercial screens. This may be due to the ability of the incomplete factorial screen to sample a broader portion of "crystallization space," using a multidimensional set of components, concentrations, and physical conditions. The incomplete factorial screen is complemented by a neural network program used to model crystallization. This capability is used to help predict new crystallization conditions. An automated, nanoliter crystallization system, with a throughput of up to 400 conditions/h in 40-nl droplets (total volume), accommodates microbatch or traditional "sitting-drop" vapor diffusion experiments. The goal of this research is to develop a fully-automated high-throughput crystallization system that integrates incomplete factorial screen and neural net capabilities.     

Recovery of carboxylic acids produced by fermentation

Carboxylic acids such as citric, lactic, succinic and itaconic acids are useful products and are obtained on large scale by fermentation. This review describes the options for recovering these and other fermentative carboxylic acids. After cell removal, often a primary recovery step is performed, using liquid–liquid extraction, adsorption, precipitation or conventional electrodialysis. If the carboxylate is formed rather than the carboxylic acid, the recovery process involves a step for removing the cation of the formed carboxylate. Then, bipolar electrodialysis and thermal methods for salt splitting can prevent that waste inorganic salts are co-produced. Final carboxylic acid purification requires either distillation or crystallization, usually involving evaporation of water.     

Automated analysis of vapor diffusion crystallization drops with an X-ray beam

Crystallogenesis, usually based on the vapor diffusion method, is currently considered one of the most difficult steps in macromolecular X-ray crystallography. Due to the increasing number of crystallization assays performed by protein crystallographers, several automated analysis methods are under development. Most of these methods are based on microscope images and shape recognition. We propose an alternative method of identifying protein crystals: by directly exposing the crystallization drops to an X-ray beam. The resulting diffraction provides far more information than classical microscope images. Not only is the presence of diffracting crystals revealed, but also a first estimation of the space group, cell parameters, and mosaicity is obtained. In certain cases, it is also possible to collect enough data to verify the presence of a specific substrate or a heavy atom. All these steps are performed without the sometimes tedious necessity of removing crystals from their crystallization drop.     

Protein isoelectric point as a predictor for increased crystallization screening efficiency

MOTIVATION: Increased efficiency in initial crystallization screening reduces cost and material requirements in structural genomics. Because pH is one of the few consistently reported parameters in the Protein Data Bank (PDB), the isoelectric point (pI) of a protein has been explored as a useful indirect predictor for the optimal choice of range and distribution of the pH sampling in crystallization trials. RESULTS: We have analyzed 9596 unique protein crystal forms from the August 2003 PDB and have found a significant relationship between the calculated pI of successfully crystallized proteins and the difference between pI and reported pH at which they were crystallized. These preferences provide strong prior information for the design of crystallization screening experiments with significantly increased efficiency and corresponding reduction in material requirements, leading to potential cost savings of millions of US$ for structural genomics projects involving high-throughput crystallographic structure determination. AVAILABILITY: A prototype example of a screen design and efficiency estimator program, CrysPred, is available at http://www-structure.llnl.gov/cryspred/     

A model-based approach for mining membrane protein crystallization trials

MOTIVATION: Membrane proteins are known to play crucial roles in various cellular functions. Information about their function can be derived from their structure, but knowledge of these proteins is limited, as their structures are difficult to obtain. Crystallization has proved to be an essential step in the determination of macromolecular structure. Unfortunately, the bottleneck is that the crystallization process is quite complex and extremely sensitive to experimental conditions, the selection of which is largely a matter of trial and error. Even under the best conditions, it can take a large amount of time, from weeks to years, to obtain diffraction-quality crystals. Other issues include the time and cost involved in taking multiple trials and the presence of very few positive samples in a wide and largely undetermined parameter space. Therefore, any help in directing scientists' attention to the hot spots in the conceptual crystallization space would lead to increased efficiency in crystallization trials. RESULTS: This work is an application case study on mining membrane protein crystallization trials to predict novel conditions that have a high likelihood of leading to crystallization. We use suitable supervised learning algorithms to model the data-space and predict a novel set of crystallization conditions. Our preliminary wet laboratory results are very encouraging and we believe this work shows great promise. We conclude with a view of the crystallization space that is based on our results, which should prove useful for future studies in this area.     

Use of novel cationic bile salts in cholesterol crystallization and solubilization in vitro

Unnatural bile salts have been synthesized with a cationic group at the side chain of natural bile acids. These cationic bile salts aggregate in water and aqueous salt solutions in a manner similar to their natural counterparts. The critical micellar concentrations of the cationic bile salts were measured using a fluorescence method. Cationic bile salts aggregated at a concentration lower than natural deoxycholic acid. Since dihydroxy bile salt micelles are well known for cholesterol dissolution/removal, the dissolution in the cationic micelles has been evaluated. The cationic analogs dissolve approximately 70 mg/dL of cholesterol, which is comparable to taurochenodeoxycholate micelle under identical bile salt concentrations. Cholesterol dissolution in cationic bile salt micelle enhanced upon adding various amounts of PC. Cholesterol crystallization was studied in model bile at various cationic bile salt concentrations. The addition of 5, 15 and 30 mM of the cationic bile salts attenuated the crystallization process, without influencing the crystal observation time or decreasing the final amount of crystals formed. All these effects were comparable to those observed with cholic acid. These findings suggest that cationic bile salts have physico-chemical properties analogous to those of natural anionic bile salts, and thus may have therapeutic potential.     

Ion hydration: Implications for cellular function, polyelectrolytes, and protein crystallization

Only oppositely charged ions with matching absolute free energies of hydration spontaneously form inner sphere ion pairs in free solution [K.D.Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311.]. We approximate this with a Law of Matching Water Affinities which is used to examine the issues of (1) how ions are selected to be compatible with the high solubility requirements of cytosolic components; (2) how cytosolic components tend to interact weakly, so that association or dissociation can be driven by environmental signals; (3) how polyelectrolytes (nucleic acids) differ from isolated charges (in proteins); (4) how ions, osmolytes and polymers are used to crystallize proteins; and (5) how the "chelate effect" is used by macromolecules to bind ions at specific sites even when there is a mismatch in water affinity between the ion and the macromolecular ligands.     

The promise of macromolecular crystallization in microfluidic chips

Microfluidics, or lab-on-a-chip technology, is proving to be a powerful, rapid, and efficient approach to a wide variety of bioanalytical and microscale biopreparative needs. The low materials consumption, combined with the potential for packing a large number of experiments in a few cubic centimeters, makes it an attractive technique for both initial screening and subsequent optimization of macromolecular crystallization conditions. Screening operations, which require a macromolecule solution with a standard set of premixed solutions, are relatively straightforward and have been successfully demonstrated in a microfluidics platform. Optimization methods, in which crystallization solutions are independently formulated from a range of stock solutions, are considerably more complex and have yet to be demonstrated. To be competitive with either approach, a microfluidics system must offer ease of operation, be able to maintain a sealed environment over several weeks to months, and give ready access for the observation and harvesting of crystals as they are grown.     

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.     

Optical resolution by preferential crystallization of 4-methylpiperidinium hydrogen (RS)-phenylsuccinate

Gibbs energy of racemate formation, binary melting point diagram, and ternary solubility diagram suggested that 4-piperidinium hydrogen (RS)-phenylsuccinate [(RS)-4-MP salt] exists in a conglomerate. Appropriate conditions were explored on the basis of free energy of critical nucleation in a supersaturated solution to resolve efficiently (RS)-4-MP salt by preferential crystallization. Successive preferential crystallization of (RS)-4-MP salt in ethanol at 20°C gave (R)- and (S)-4-MP salts of 90–94% optical purities. Optically pure (R)- and (S)-phenylsuccinic acids were obtained by recrystallization of the (R)- and (S)-4-MP salts, followed by treatment of the salts purified with hydrochloric acid. © 1994 Wiley-Liss, Inc.     

Influence of precipitating agents on thermodynamic parameters of protein crystallization solutions

X‐ray crystallography is the most powerful method for determining three‐dimensional structures of proteins to (near‐)atomic resolution, but protein crystallization is a poorly explained and often intractable phenomenon. Differential Scanning Calorimetry was used to measure the thermodynamic parameters (ΔG, ΔH, ΔS) of temperature‐driven unfolding of two globular proteins, lysozyme, and ribonuclease A, in various salt solutions. The mixtures were categorized into those that were conducive to crystallization of the protein and those that were not. It was found that even fairly low salt concentrations had very large effects on thermodynamic parameters. High concentrations of salts conducive to crystallization stabilized the native folded forms of proteins, whereas high concentrations of salts that did not crystallize them tended to destabilize them. Considering the ΔH and TΔS contributions to the ΔG of unfolding separately, high concentrations of crystallizing salts were found to enthalpically stabilize and entropically destabilize the protein, and vice‐versa for the noncrystallizing salts. These observations suggest an explanation, in terms of protein stability and entropy of hydration, of why some salts are good crystallization agents for a given protein and others are not. This in turn provides theoretical insight into the process of protein crystallization, suggesting ways of predicting and controlling it. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 642–652, 2016.     

Protein crystallization in the structural genomics era

There are five broad areas where noteworthy advances have occurred in the field of macromolecular crystallization in the past 10 years, though some areas have seen the major part of those advances in only the last two years. This is largely a consequence of the international structural genomics initiative and its early results. The five areas are: (1) Physical studies and characterization of the protein crystallization process; (2) Development of new practical approaches and procedures; (3) The implementation of protein engineering by genetic means to enhance both purification and crystallization; (4) The creation of new screening conditions based on information and databases emerging from structural genomics; and (5) Development and implementation of automation, robotics, and mass screening of crystallization conditions using very small amounts of protein. A brief summary is provided here of the progress in the past few years and the influence of the structural genomics project.     

Statistical analysis of 15 dimensions in the crystallization space for protein-DNA complexes

Solving the three dimensional structure of a protein-DNA complex is a prerequisite to understand, at the atomic level, the interactions between DNA-binding proteins and their target DNA sequences. Arranging these complexes into an ordered and repetitive network (a crystal, suitable for X-Ray analysis) is a time-limiting empirical step. Although it has been suggested that the crystallization space for protein-DNA complexes is probably smaller than that of non-complexed proteins, a study presenting a detailed and updated analysis of this space is still missing. Here, we analyze the successful crystallization conditions of several hundred protein-DNA complexes and present a bias-free statistical analysis of 15 crystallization parameters that include concentration, temperature, pH, precipitants, salts, divalent cations and polyamines. Our analysis shows that some crystallization parameters are interestingly restricted into narrow intervals. These restrictions could be very helpful in the design of sparse-matrix crystallization screens that target exclusively protein-DNA complexes.     

Data mining crystallization databases: knowledge-based approaches to optimize protein crystal screens

Protein crystallization is a major bottleneck in protein X-ray crystallography, the workhorse of most structural proteomics projects. Because the principles that govern protein crystallization are too poorly understood to allow them to be used in a strongly predictive sense, the most common crystallization strategy entails screening a wide variety of solution conditions to identify the small subset that will support crystal nucleation and growth. We tested the hypothesis that more efficient crystallization strategies could be formulated by extracting useful patterns and correlations from the large data sets of crystallization trials created in structural proteomics projects. A database of crystallization conditions was constructed for 755 different proteins purified and crystallized under uniform conditions. Forty-five percent of the proteins formed crystals. Data mining identified the conditions that crystallize the most proteins, revealed that many conditions are highly correlated in their behavior, and showed that the crystallization success rate is markedly dependent on the organism from which proteins derive. Of the proteins that crystallized in a 48-condition experiment, 60% could be crystallized in as few as 6 conditions and 94% in 24 conditions. Consideration of the full range of information coming from crystal screening trials allows one to design screens that are maximally productive while consuming minimal resources, and also suggests further useful conditions for extending existing screens.     

Searching for silver bullets: an alternative strategy for crystallizing macromolecules

Based on a hypothesis that various small molecules might establish stabilizing, intermolecular, non covalent crosslinks in protein crystals and thereby promote lattice formation, we carried out three separate experiments. We assessed the impact of 200 chemicals on the propensity of 81 different proteins and viruses to crystallize. The experiments were comprised of 18240 vapor diffusion trials. A salient feature of the experiments was that, aside from the inclusion of the reagent mixes, only two fundamental crystallization conditions were used, 30% PEG 3350, and 50% Tacsimate at pH 7. Overall, 65 proteins (85%) were crystallized. Most significant was that 35 of the 65 (54%) crystallized only in the presence of one or more reagent mixes, but not in control samples lacking any additives. Among the most promising types of reagent mixes were those composed of polyvalent, charged groups, such as di and tri carboxylic acids, diamino compounds, molecules bearing one or more sulfonyl or phosphate groups, and a broad range of common biochemicals, coenzymes, biological effectors, and ligands. We propose that an alternate approach to crystallizing proteins might be developed, which employs a limited set of fundamental crystallization conditions combined with a broad screen of potentially useful small molecule additives.     

Taste-mediated behavioral and electrophysiological responses by the predatory fish Ariopsis felis to deterrent pigments from Aplysia californica ink

Chemical defenses are used by many organisms to avoid predation, and these defenses may function by stimulating predators’ chemosensory systems. Our study examined detection mechanisms for components of defensive ink of sea hares, Aplysia californica, by predatory sea catfish, Ariopsis felis. Behavioral analyses show aplysioviolin and phycoerythrobilin are detected intra-orally and by barbels and are deterrent at concentrations as low as 0.1% full strength. We performed electrophysiological recordings from the facial–trigeminal nerve complex innervating the maxillary barbel and tested aplysioviolin, phycoerythrobilin, amino acids, and bile salts in cross-adaptation experiments. Amino acids and bile salts are known stimulatory compounds for teleost taste systems. Our results show aplysioviolin and phycoerythrobilin are equally stimulatory and completely cross-adapt to each other’s responses. Adaptation to aplysioviolin or phycoerythrobilin reduced but did not eliminate responses to amino acids or bile salts. Adaptation to amino acids or bile salts incompletely reduced responses to aplysioviolin or phycoerythrobilin. The fact that cross-adaptations with aplysioviolin and phycoerythrobilin were not completely reciprocal indicates there are amino acid and bile salt sensitive fibers insensitive to aplysioviolin and phycoerythrobilin. These results indicate two gustatory pathways for aplysioviolin and phycoerythrobilin: one independent of amino acids and bile salts and another shared with some amino acids.     

Protein crystallization by design: chymotrypsinogen without precipitants

Protein crystals are usually obtained by an empirical approach based on extensive screening to identify suitable crystallization conditions. In contrast, we have used a systematic predictive procedure to produce data-quality crystals of bovine chymotrypsinogen A and used them to obtain a refined X-ray structure to 3 A resolution. Measurements of the osmotic second virial coefficient of chymotrypsinogen solutions were used to identify suitable solvent conditions, following which crystals were grown for approximately 30 hours by ultracentrifugal crystallization, without the use of any precipitants. Existing structures of chymotrypsinogen were obtained in solutions including 10-30 % ethanol, whereas simple buffered NaCl solutions were used here. The protein crystallized in the tetragonal space group P4(1)2(1)2, with one molecule per asymmetric unit. The quality of the refined map was very high throughout, with the main-chain atoms of all but four residues clearly defined and with nearly all side-chains also defined. Although only minor differences are seen compared to the structures previously reported, they indicate the possibility of structural changes due to the crystallization conditions used in those studies. Our results show that more systematic crystallization of proteins is possible, and that the procedure can expand the range of conditions under which crystals can be grown successfully and can make new crystal forms available.     

Online monitoring of preferential crystallization of enantiomers

Polarimetry is used for continuous online monitoring of optical resolution by preferential crystallization. In combination with refractometry the liquid phase composition is determined, allowing one to follow the resolution progress quantitatively. The measurement techniques were calibrated up to relatively high solution concentrations and combined with the crystallizer. The resolution of DL-threonine was performed by preferential crystallization experiments in aqueous solution varying several process parameters like supersaturation, seed amount, initial enantiomeric excess, and scale. The resolution progress can be conveniently described by profiles of the optical rotation (polarimetric signal) and the crystallization pathway in the corresponding ternary phase diagram. The method outlined is applicable for dynamic process optimization and control purposes in "quasi-continuous" chiral separation processes.     

Precipitants and additives for membrane crystallization of lysozyme

Membrane crystallization is a newly developed crystallization technique that has proven to be superior in producing good crystal forms under operating conditions that are not appropriate to perform the crystallization process by other traditional techniques. In this work, static membrane crystallization was carried out on lysozyme, with hollow-fiber microporous hydrophobic membranes. Numerous precipitant and additive types and concentrations were employed in the crystallization processes in order to select the most appropriate precipitant and additive types and to find their corresponding concentration levels that can yield the best crystal forms. The crystallization processes were analyzed in two ways: firstly, by evaluation of the transmembrane fluxes obtained by using different precipitants and additives; secondly, by utilization of the images and results obtained from the micrography and IR spectra in comparisons and evaluations of the crystals formed under all kinds of conditions. Moreover, the size distributions of the crystals yielded under several typical crystallization conditions were analyzed, and turbidity and induction time periods obtained during typical crystallization experiments were also measured. Amongst the numerous precipitants and additives tested, the most appropriate precipitant type and additive were chosen and their concentrations were optimized. Good lysozyme crystals were obtained using a certain precipitant and additive. The obtained results from this work further support the advantages of utilizing the membrane crystallization technique for macromolecule crystallizations.     

Effects of strong electrolytes on the iron alum-Alcian Blue-Safranin staining of mast cell granules of the rat

Summary Rat mast cells fixed in Carnoy's fluid were stained with iron alum-Alcian Blue-Safranin solution after pre-treatment with strong electrolyte solutions including acids, neutral salts and alkalis. Although both red and blue mast cells were observed without pre-treatment, most mast cells were stained blue and a few red when they were stained after the pre-treatment. Mast cell granules contain salt complexes formed between basic proteins and acidic polysaccharides through ionic linkages between protein basic groups and polysaccharide sulphate and carboxylic acid groups. It is suggested that when sections are treated with strong electrolyte solutions, complexes are broken by disruption of ionic linkages and sulphate and carboxylic acid groups of polysaccharides masked by basic proteins become available for binding Alcian Blue. This was confirmed by model experiments performed with smears of a heparin-lysozyme complex.When mast cells were fixed in aldehyde-containing fixatives, no effects of strong electrolyte solutions on the staining properties of mast cell granules were revealed.