Protein Crystal Growth Research Articles

Overcoming merohedral twinning in crystals of bacteriorhodopsin grown in lipidic mesophase

Twinning is one of the most common crystal-growth defects in protein crystallography. There are neither efficient rational approaches for the growth of nontwinned protein crystals nor are there examples of systematic studies of the dependence of the twinning-ratio distribution on crystallization conditions. The description of the twinning phenomenon has been covered even less for membrane-protein crystals and is non-existent for crystals grown using lipidic phases (in meso). In the present work, possibilities for overcoming merohedral twinning are investigated for crystals of the membrane protein bacteriorhodopsin (bR) grown in meso. It is shown that traditional crystallization additives are not effective in the case of the in meso crystallization of bR. The twinning ratio was determined for 310 crystals grown under different crystallization conditions. A correlation of the twinning ratio with the growth rate of the crystals was observed. Slow growth indicated that crystals had a noticeable chance of avoiding twinning. Model calculations were performed in order to rationalize this observation. The calculations confirmed the experimental observation that most crystals consist of two twin domains and showed that under this condition small changes in the probability of twin-domain formation lead to dramatic changes in the number of nontwinned crystals, which explains why slow crystal growth results in a considerable number of nontwinned crystals.

Channel height dependent protein nucleation and crystal growth in microfluidic devices

We report on results of a study of protein crystallization in microfluidic devices with different channel heights. Multilayer soft lithography has been used for the fabrication of devices with integrated micro-valves and crystallization channels of height in the range from 15 μm to 180 μm. To demonstrate the channel height dependent nucleation and crystal growth, a standard batch crystallization solution composed of 60 mg/ml lysozyme, 100 mM acetate buffer pH 4.6 and 1.5 M NaCl was used with minimized sample quantity. Our results show that deep channels favorite the nucleation whereas shallow ones favorite the crystal growth. When the channel height is less than 50 μm the number of lysozyme crystals is dramatically reduced whereas their mean size is increased. Furthermore, our results also show the feasibility of decoupling nucleation and crystal growth in a stair-like channel which should facilitate the appearance of single crystals suitable for X-ray diffraction.

Growth of Large Protein Crystals by Top-Seeded Solution Growth Together with the Floating and Solution-Stirring Technique

Neutron protein crystallography is expected to yield a more precise understanding of the protein structure by visualizing hydrogen atoms and hydration water molecules. However, relatively few protein structures have been analyzed by neutron diffraction, in part because very large crystals of the target proteins are required in order to compensate for the weak flux of the available neutron beam. Here, we report the crystal growth of hen egg white lysozyme (HEWL) by the top-seeded solution growth with the floating and solution-stirring method (TSSG-FAST), the first utilized for protein crystallization. A series of crystal-growth experiments demonstrated that TSSG-FAST is an efficient strategy for rapidly obtaining large single crystals and effectively preventing polycrystallization of the seed crystal. Furthermore, the shape of the HEWL crystals was found to be strongly influenced by the orientation of the seed crystal, suggesting that the shape of the HEWL crystal may be controlled by TSSG-FAST. It is anticipated that this new approach will expand the limits on the growth of large protein crystals for neutron protein crystallography.

Protein crystal growth on the Russian segment of the International Space Station

Experiments on protein crystallization on the Russian segment of the International Space Station were started in 2005. These experiments were performed in the Modul’-1 protein crystallization apparatus specially designed for crystal growth by the free-interface-diffusion method. This paper describes experiments on the crystallization of lysozyme, carboxypeptidase B, and recombinant human insulin on Earth and in microgravity using the Modul’-1 protein crystallization apparatus during the ISS-11-ISS-14 space flights. Crystals of all proteins grown in microgravity have larger sizes than those grown on Earth. Space-grown crystals of lysozyme and insulin characterized by X-ray diffraction were shown to diffract to higher resolution than the Earth-grown crystals. The three-dimensional structures of Zn-insulin crystals grown both on Earth and in microgravity were established. The conformation of the Zn-insulin hexamer in the crystalline state is described.

Layered crystals of apo‐ and holoferritin grown by alternating crystallization

AbstractWe report on the use of alternating crystallization for deposition of layers of different (though closely related) proteins in a single crystal. Investigations were carried out with the unique protein couple consisting of two forms of ferritin, apoferritin and holoferritin from horse spleen, which, despite being of quite different molecular masses, still possess identical organic shells. Crystals of both proteins were used as substrates for subsequent contiguous growth of the partner protein in perfect alignment. We observed continuous growth of combined (onion‐like) single crystals; artificial structures of biological macromolecules can be designed in this way. The homoepitaxial layered growth shows in an unambiguous way that protein crystallization depends only on the surface protein conformation and amino‐acid composition, but not on the internal molecule structure. The limitations of protein crystal growth for designing layered structures of biological macromolecules were revealed by growing of heterogeneous protein crystals onto pre‐existing protein crystalline substrates. Tetragonal crystals of hen egg‐white lysozyme were grown onto cubic apoferritin crystals used as substrates. It was observed that the lysozyme crystals were not lattice‐matched to the ‘host’ apoferritin crystals; this led to mere aggregates of different crystals. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

A Synthetic Protein Selected for Ligand Binding Affinity Mediates ATP Hydrolysis

How primitive enzymes emerged from a primordial pool remains a fundamental unanswered question with important practical implications in synthetic biology. Here we show that a de novo evolved ATP binding protein, selected solely on the basis of its ability to bind ATP, mediates the regiospecific hydrolysis of ATP to ADP when crystallized with 1 equiv of ATP. Structural insights into this reaction were obtained by growing protein crystals under saturating ATP conditions. The resulting crystal structure refined to 1.8 A resolution reveals that this man-made protein binds ATP in an unusual bent conformation that is metal-independent and held in place by a key bridging water molecule. Removal of this interaction using a null mutant results in a variant that binds ATP in a normal linear geometry and is incapable of ATP hydrolysis. Biochemical analysis, including high-resolution mass spectrometry performed on dissolved protein crystals, confirms that the reaction is accelerated in the crystalline environment. This observation suggests that proteins with weak chemical reactivity can emerge from high affinity ligand binding sites and that constrained ligand-binding geometries could have helped to facilitate the emergence of early protein enzymes.

The development of biochemistry and molecular biology in China

The development of biochemistry and molecular biology in China

A general protocol for the crystallization of membrane proteins for X-ray structural investigation

Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.

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.

Control of Nucleation Rate for Tetragonal Hen-Egg White Lysozyme Crystals by Application of an Electric Field with Variable Frequencies

Control of the nucleation rate is important for the growth of high quality protein crystals; therefore, the development of improved growth methods is required. Crystal growth with an external electric field is an attractive method for control of the nucleation rate. However, mainly a decrease in nucleation rate was observed when an external electric field was applied using DC voltage. By focusing on the chemical potential of the liquid and solid, control of both the increase and the decrease in nucleation rate was successfully achieved by applying an AC electric field. Control of nucleation rate in terms of enhancement and retardation is reported for hen-egg white lysozyme crystals by imposition of an electric field with varying frequencies, which was determined by the electrical permittivity of both the hen-egg white lysozyme solution and crystals.

Measuring the Nucleation Rate of Lysozyme using Microfluidics

We employ the PhaseChip, a (poly)dimethylsiloxane (PDMS) microfluidic device, for statistical studies of protein crystal nucleation. The PhaseChip is designed to decouple nucleation and growth of protein crystals and so improve their yield and quality. Two layers of fluidic channels containing salt reservoirs and nanoliter-sized wells for protein drops in oil are separated by a thin PDMS membrane, which is permeable to water, but not to salt or macromolecules such as protein. We reversibly vary the supersaturation of protein inside the stored droplets by controlling the chemical potential of the reservoir. Lysozyme in the presence of sodium chloride is used as a model system. We determine the crystal nucleation rate as a function of protein supersaturation by counting the number of crystal nuclei per droplet, as demonstrated by Galkin and Vekilov.1.

Flow suspended solution growth—A new method

We report on the development of a new method of crystal growth from the solution in which the growing crystal is freely suspended in the flowing mother solution. We call this method flow-suspended solution growth. Growth of the crystal is possible without any wall contact in up-flow of supersaturated solution in a conical vertical tube called growth reactor. The velocity profile of the solution in the tube around the growing crystal centres the object on the axis of the tube. This is possible under certain limitations of the crystal size and of the density difference Δ ρ between crystal and solution. We describe and discuss these limitations. The method was successfully tested with solution growth of KAl(SO 4) 2·12H 2O from watery solution. We report some practical information like the relationships between crystal size, Δ ρ, diameter of the growth reactor and the needed volume flow rate to suspend the crystal. We report the possibility of long-time unattended steady growth and an easy seeding and harvesting action. Flow-suspended solution growth has the potential for controlled protein crystal growth (if miniaturized) and for the growth of biological objects like embryos.

Simple Geometry for Diffusion Limited Protein Crystal Growth: Harnessing Gravity to Suppress Convection

A new upside-down geometry is proposed to achieve the beneficial effects of microgravity crystal growth by making use of buoyant forces instead of compensating for them. We show by growth experiments on sodium chlorate and lysozyme that crystal growth in an upside-down geometry leads to the formation of a buoyancy assisted depletion zone where convection is suppressed. The effects on growth rate and morphology that are observed are all indicative of diffusion limited growth, just as would happen in the absence of gravity. The optical quality of the lysozyme crystals clearly improved, but no effects of the growth method on X-ray diffraction quality could be observed. The simplicity of the growth geometry offers the possibility to perform large scale protein crystal growth experiments under microgravity-like conditions, without the requirement of compensating for gravity.

Buoyant plumes from solute gradients generated by non-motile Escherichia coli

The effect of hydrodynamic mixing in bacterial populations due to bacterial chemotaxis is a well-described phenomenon known as bioconvection. Here we report the observation of buoyant plumes that result in hydrodynamic mixing, but in contrast to bioconvection the plumes form in the absence of bacterial motility. We propose that the buoyant flow originates from solute gradients created by bacterial metabolism, similar to solute-induced buoyant flow around growing protein crystals. In our experiments, metabolically-active non-motile Escherichia coli were layered along the bottom of flat-bottomed containers. The E. coli consumed glucose in the medium creating a lighter fluid beneath a heavier fluid. The situation is an example of Rayleigh–Taylor instability, in which a lighter fluid pushes on a heavier one. We developed a numerical model to study the effect of E. coli nutrient consumption and by-product excretion on extracellular solute gradients. The model solutions showed reduced-density fluid along the bottom of the fluid domain leading to buoyant plumes, which were qualitatively similar to the experimental plumes. We also used scaling analyses to study the dependence of plume formation on container size and cell size, and to investigate the effect of reduced gravity, such as the microgravity conditions encountered during spaceflight.

Age-Dependent Evolution of the Yeast Protein Interaction Network Suggests a Limited Role of Gene Duplication and Divergence

Proteins interact in complex protein–protein interaction (PPI) networks whose topological properties—such as scale-free topology, hierarchical modularity, and dissortativity—have suggested models of network evolution. Currently preferred models invoke preferential attachment or gene duplication and divergence to produce networks whose topology matches that observed for real PPIs, thus supporting these as likely models for network evolution. Here, we show that the interaction density and homodimeric frequency are highly protein age–dependent in real PPI networks in a manner which does not agree with these canonical models. In light of these results, we propose an alternative stochastic model, which adds each protein sequentially to a growing network in a manner analogous to protein crystal growth (CG) in solution. The key ideas are (1) interaction probability increases with availability of unoccupied interaction surface, thus following an anti-preferential attachment rule, (2) as a network grows, highly connected sub-networks emerge into protein modules or complexes, and (3) once a new protein is committed to a module, further connections tend to be localized within that module. The CG model produces PPI networks consistent in both topology and age distributions with real PPI networks and is well supported by the spatial arrangement of protein complexes of known 3-D structure, suggesting a plausible physical mechanism for network evolution.

On Electrochemically Assisted Protein Crystallization and Related Methods

In this communication, the basic strategies to control the kinetics and transport phenomena of the crystal growth process, as well as novel methods to induce either nucleation or protein crystal growth via electrochemical processes, are reviewed. Additionally, recent advances, where low direct/alternant current or voltages are applied to the crystal growth cell to obtain high-quality single crystals for X-ray diffraction, are also discussed.

The role of solutal convection in protein crystal growth—A new dimensionless number to evaluate the effects of convection on protein crystal growth

According to the experiments by Vekilov et al., the crystal growth rate, G c of tetragonal lysozyme crystal initially increases up to 200 μm/s with increasing flow velocity, u m and then decreases monotonically. While in the lower supersaturated solution, G c decreases monotonically with increasing u m, and the growth cessation occurs at u m=1450 μm/s. In this paper, we propose a new theory to explain these complicated experimental results. We consider one protein molecule near the crystal surface and define a dimensionless number r, the ratio of the velocity of convective flow to the diffusion velocity of a protein molecule. When convection occurs along the crystal surface and a molecule diffuses toward the crystal surface, r indicates the moving direction of the molecule and is closely related with crystal growth rate. We derive the equation to calculate r from experimental parameters. Analyzing the experimental results in the literatures, we find that the dependence of G c on u m is related with r and there exist three critical r-values, r 1≈4–8, r 2≈0.01–0.02, r 3≈0.004. When r> r 1, crystal growth cession occurs; when r 1> r> r 2, G c decreases with u m; when r 2> r> r 3, G c increases with u m; when r< r 3, G c is independent of u m. Though our model is very simple, the dimensionless number r can explain the effects of convection on crystal growth rate consistently.

Influence of micro-impurity on protein crystal growth studied by the etch figure method

The investigation of the effect of micro impurity on crystal growth by optical microscopy has been validated. The results showed that the growth rate of a lysozyme crystal was affected even if the concentration of impurity of fluorescent-labeled lysozyme (abbreviation, F-lysozyme) was very small. Different concentrations of F-lysozyme had different effects on crystal growth rate. The growth rate decreased much more as F-lysozyme concentration increased. The density of incorporated F-lysozyme on different grown layers of a lysozyme crystal during crystal growth was obtained from the results of flat-bottomed etch pits density.

Growth of large protein crystals for neutron crystallography by hanging a seed crystal

Growth of large protein crystals for neutron crystallography by hanging a seed crystal

The Critical Rayleigh Number in Low Gravity Crystal Growth from Solution

If crystal growth from solution takes place in a closed container, a critical Rayleigh number can be defined, below which buoyancy driven convection is suppressed and mass transport is completely determined by diffusion at gravitational accelerations higher than 0g. Using finite element simulations and an analytical model, we show that it is possible to predict the critical value of the Rayleigh number. This result can be used to optimize growth conditions for microgravity protein crystal growth, if the gravitational acceleration cannot be canceled completely, like in space, or is canceled inhomogeneously, like in gradient magnetic fields.