What is a protein crystal? Can we apply the terminology of classical industrial crystallization to them?

The nature of protein crystals is discussed. Since protein crystals contain not only proteins but also other substances the usage of conventional terms (of industrial crystallization) of naming such crystals is questioned. The proof that there are other components than the protein itself in a protein crystal is given. It is suggested to use a procedure like in biotechnology to guarantee the production of the right protein crystals according to the PAT concept. It is suggested not to use other established names like “hydrates”, “solvates”, “but crystals for proteins since the definitions do not fit the nature of the protein crystals.     

Total external reflection X-ray fluorescence analysis of protein-metal ion interactions in biological systems

This paper presents the results of an investigation into hemoglobin-based protein films that were formed on a liquid surface. X-ray standing wave measurements were performed at the ID 10 beamline of the European Synchrotron Radiation Facility (ESRF) and at the Langmuir station of the Kurchatov Synchrotron Radiation Source. It was found that the ability of the protein to bind metal ions is substantially increased due to the conformational rearrangements of protein macromolecules caused by various damaging effects. The elemental composition of protein preparations, which were isolated from children and adults with chronic metabolic diseases accompanied by endogenous intoxication, was analyzed. The results of the investigations offer evidence that an increase in the ligand-binding properties of the protein molecules, which was observed in model experiments using protein films, is a common trait and corresponds to in vivo processes accompanying metabolic disturbances in the body.     

Protein crystal nucleation: Recent notions

The nucleation of protein crystals is reconsidered taking into account the specificity of the protein molecules. In contrast to the homogeneous surface properties of small molecules, the protein molecule surface is highly inhomogeneous. Over their surfaces proteins exhibit high anisotropic distribution of patches, which are able to form crystalline bonds, the crystallization patch representing only a small fraction of the total surface of the protein molecule. Therefore, an appropriate spatial orientation of the colliding protein molecules is required in order to create a crystalline cluster. This scenario decreases considerably the success ratio of the attempt frequency for crystal nucleation. On the other hand a heterogeneous nucleation of (protein) crystals may be accelerated due to the arrival on some support of under‐critical clusters that are formed in bulk solution; when arriving there they may acquire the property of critical nuclei. Thus, a plausible explanation of important peculiarities of protein crystal nucleation, as inferred from the experimental data, is suggested. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Influence of various factors on the liquid/liquid diffusion crystallization of proteins

From a numerical simulation of pre-nucleation solute transport in the liquid/liquid diffusion crystallization of proteins, it is derived that there are various factors influencing the spatial and temporal distributions of supersaturation. They include the ratio of the length of the protein solution to that of the salt solution in a tube crystallizer, the initial concentrations of protein and salt, and the initial salt concentration in the protein solution. Their influences on the protein crystallization have been demonstrated by the corresponding experiments of gel crystal growth. It may be deduced that the experimental conditions for the liquid/liquid diffusion growth of protein crystals could be optimized under given conditions in order to develop the advantages of this method.     

The effect of diluting crystallization droplets on protein crystallization in vapor diffusion method

In this study, effects of diluting either protein or crystallization agents in the droplets on the success rate of protein crystallization was investigated. Diluting the crystallization agent was found to increase the success rate of protein crystallization. Theoretical analysis showed that, concentration ranges of both protein and crystallization agent that can be scanned during the vapor diffusion process are wider with diluting the crystallization agent than that without dilution, resulting in more opportunities for the crystallization solution to be in the nucleation zone. On the other hand, diluting protein could lead to controversial results depending on the location of the initial concentration relative to that of the nucleation zone in the phase diagram. The method of diluting the crystallization agent is therefore proposed as an alternative modification to the conventional vapor diffusion method for obtaining more crystallization conditions in protein crystallization screening. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Small-angle X-ray scattering study of conditions for the formation of growth units of protein crystals in lysozyme solutions

The structural composition of lysozyme solutions favorable for the formation of the tetragonal form of protein crystals was studied by synchrotron-based small-angle X-ray scattering depending on the protein concentration and the temperature. Along with lysozyme monomers, dimers and octamers are found in crystallization solutions; the octamer content increases with an increase in the protein concentration.     

Modification of the Langmuir–Schaefer method for fabrication of ordered protein films

A modification of the Langmuir–Schaefer method for the fabrication of high-quality protein films on a solid substrate was proposed and applied to lysozyme. The procedure relies on the use of a pre-prepared protein solution, the parameters of which correspond to crystallization conditions. A lysozyme Langmuir monolayer was shown to be formed with the involvement of complexes, namely, dimers and octamers of protein molecules that are present in such protein solutions. These complexes apparently retain the structure after spreading a protein solution onto an aqueous subphase in a Langmuir trough. The thickness of the film after the transfer of the monolayer, which was formed by the proposed procedure, onto a solid substrate corresponds to the diameter of the octamer and this film is dense, continuous, and uniform, as was demonstrated by several methods: X-ray reflectivity, total external reflection X-ray standing wave, and atomic force microscopy. A layer of chloride ions that formed under the Langmuir monolayer was found at the air–protein film interface. This fact confirms an important role of the precipitating agent (chloride ions) in all steps of the formation of lysozyme films.     

Nucleation of protein crystals

Protein crystal nucleation is a central problem in biological crystallography and other areas of science, technology, and medicine. Recent studies have demonstrated that protein crystal nuclei form within crucial precursors. Data for several proteins provided by these methods have demonstrated that the nucleation precursors are clusters consisting of protein dense liquid, which are metastable with respect to the host protein solution. The clusters are several hundred nanometers in size, they occupy from 10⁻⁷ to 10⁻³ of the solution volume, and their properties in solutions supersaturated with respect to crystals are similar to those in homogeneous, i.e., undersaturated, solutions. The clusters exist due to the conformation flexibility of the protein molecules, leading to the exposure of hydrophobic surfaces and enhanced intermolecular binding. These results indicate that protein conformational flexibility might be the mechanism behind the metastable mesoscopic clusters and crystal nucleation. The investigations of the cluster properties are still in their infancy. Results on direct imaging of cluster behaviors and characterization of cluster mechanisms with a variety of proteins will soon lead to major breakthroughs in protein biophysics.     

Layered crystals of apo‐ and holoferritin grown by alternating crystallization

We 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)     

Supersaturation patterns in counter-diffusion crystallisation methods followed by Mach–Zehnder interferometry

We present experimental observation of the spatio-temporal pattern of supersaturation in counter-diffusion methods. These complex patterns were recorded by dynamical interferometric analysis using a Mach–Zehnder configuration. Tetragonal hen egg white lysozyme crystals were grown inside APCF (advanced protein crystallisation facility) reactors. Salt and protein diffusion profiles were obtained independently by performing duplicated experiments with and without protein in the protein chamber; salt gradients were observed directly while protein concentration profiles are computed from the differences in refractive index between the two experiments. As expected from computer simulations, the time evolution of supersaturation shows a maximum about 45 h after activation (although this value can change as a function of the starting conditions and the geometry of the reactor). Nucleation takes place before this maximum supersaturation is reached. This explains the trend of the growth rate versus time curves for experiments performed within APCF reactors (both on ground and in space) and in capillaries by the gel acupuncture technique. By using very low concentration agarose gel in the protein chamber, sedimentation and buoyancy effects are eliminated so that the effects of gravity on fluid dynamics and hence on the spatio-temporal evolution of supersaturation can be assessed. These results confirm experimentally the predicted behaviour of counter-diffusion systems and support their use in growing large high-quality protein single crystals.     

Heterogeneous versus bulk nucleation of lysozyme crystals

Heterogeneous (on‐glass) protein crystal nucleation was separated from the bulk one in systems of thin protein solution layers, confined between two glass plates of custom made quasi two‐dimensional all‐glass cells, as well as by applying forced protein solution flow. Two commercial samples of hen‐egg‐white lysozyme, Seikagaku and Sigma were used as model proteins. Applying the classical technique of separation in time of nucleation and growth stages with protein solution layers of thickness 0.05 cm we found that the on‐glass crystal nucleation prevailed highly with Seikagaku HEWL, while on the opposite, bulk nucleated crystals represented the main crystal fraction in Sigma solution. Also using 0.05 cm solution layers nucleation rates were measured separately for the on‐glass and bulk protein crystals. The process was investigated by varying solution layer thicknesses as well, from 0.05 down to 0.01, 0.0065 and 0.002 cm. Studying the influence of the forced protein solution flow on HEWL crystal nucleation the classical double‐pulse technique was modified by separating the nucleation and growth stages not only in time, but simultaneously also in place. In this case we found that the ratio of on‐glass formed crystal nuclei to bulk nuclei depended on the flow velocity, but in different manner with Seikagaku HEWL and Sigma HEWL. A plausible explanation of our experimental results is that the bulk crystal nucleation occurs on foreign surfaces as well, e.g. on rests of source biomaterial, which are always present in the protein solutions. Moreover, biomaterial seems to be more active nucleant than glass. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

X-ray fluorescence studies for the elemental composition and molecular organization of protein films on the surface of the liquid subphase

This paper reports on the results of the investigation of protein films that are based on alkaline phosphatase and glucose oxidase enzymes and formed on the surface of the liquid subphase. The experimental studies have been performed using total external reflection X-ray fluorescence spectrometry at the European Synchrotron Radiation Facility (Grenoble, France). The self-organization processes that occur in protein systems on the surface of the liquid subphase under the conditions where the protein molecules retain their mobility have been investigated using X-ray fluorescence measurements for the first time.     

Development of an Automated High Throughput LCP-FRAP Assay to Guide Membrane Protein Crystallization in Lipid Mesophases

Crystallization in lipidic mesophases (in meso) has been successfully used to obtain a number of high-resolution membrane protein structures including challenging members of the human G protein-coupled receptor (GPCR) family. Crystallogenesis in arguably the most successful mesophase, lipidic cubic phase (LCP), critically depends on the ability of protein to diffuse in the LCP matrix and to form specific protein-protein contacts to support crystal nucleation and growth. The ability of an integral membrane protein to diffuse in LCP is strongly affected by the protein aggregation state, the structural parameters of LCP, and the chemical environment. In order to satisfy both requirements of diffusion and specific interactions, one must balance multiple parameters, such as identity of LCP host lipid, composition of precipitant solution, identity of ligand, and protein modifications. Screening within such multi-dimensional crystallization space presents a significant bottleneck in obtaining initial crystal leads. To reduce this combinatorial challenge, we developed a pre-crystallization screening assay to measure the diffusion characteristics of a protein target in LCP. Utilizing the Fluorescence Recovery After Photobleaching (FRAP) technique in an automated and high throughput manner, we were able to map conditions that support adequate diffusion in LCP using a minimal amount of protein. Data collection and processing protocols were validated using two model GPCR targets: the β(2)-adrenergic receptor and the A(2A) adenosine receptor.     

Brittleness of protein crystals

A simple technique for studying the brittleness of small crystals is reported. The limits of fracture toughness of tetragonal hen‐egg white lysozyme crystals, oriented with their c‐axis normally to the substrate, were measured. The strong mechanics anisotropy of those crystals was confirmed. The role of the water present in the protein crystal lattice was re‐considered in seek for a more holistic understanding of the process, the idea being that the intra‐crystalline solution sustains the globular protein molecules in their native configuration. Also it is argued that this water may contribute for holding together the huge bio‐molecules in the crystal lattice (that is to act as additional “glue” in the crystal). The hypothesis is that dynamic chains of H‐bonds in the intra‐crystalline water are likely to be prolonged to connect protein‐to‐water‐to‐protein.     

Small-Angle X-ray Scattering Study of Macrophage Migration Inhibitory Factor Complexed with Albumin

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, which plays a pivotal role in the regulation of immune response. Hence, the search for new inhibitors of MIF tautomerase activity has attracted great attention. This protein is known to serve as a superligand, by involving in protein–protein interactions that are poorly studied. Macrophage migration inhibitory factor was prepared in complex with albumin, and its solution structure was studied. Difficulties encountered in performing this research were due to the fact that the sample was a mixture of the MIF–albumin complex and the individual proteins. The interaction was found to be weak and unstable. Three most probable models of the MIF–albumin complex were obtained using small-angle X-ray scattering and molecular docking simulation, and one of these models was shown to be preferable. One albumin molecule binds to the MIF trimer in the active-site region of the protein.     

Correlating dynamic amino acid properties with success rate of crystallization of proteins from Bacteroides vulgatus

To enhance the success rate of protein crystallization, many studies were conducted to determine the relationship between amino acid properties and the success rate of protein crystallization. Although those were successful, new efforts should be made to search for the new factors, which affect protein crystallization. In this study, two dynamic amino acid properties were used to correlate with the success rate of crystallization of proteins from Bacteroides vulgatus, because the amino acid properties used in previous studies were steady. As previously done, logistic regression and neural network were used to model that relationship, and the results were compared against those obtained from each of 532 amino acid properties, which severed as benchmark. The results demonstrated that dynamic amino acid properties should be taken into consideration of protein crystallization. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Estimated effects of silicone glue on protein crystal growth

Silicone glue (modified silicone polymer) is widely used for both experiments involving inorganic crystal growth and those involving organic materials like proteins. This material is very useful for building a hand-made experiment setup or for fixing protein crystals to specific locations. Though silicone glue is regarded as harmful to proteins, no systematic verification was performed to investigate its impurity effects on protein crystal growth. We focused on and estimated the impurity effects of silicone glue on protein crystal growth.     

Bond selection during protein crystallization: Crystal shapes

The so‐called bond selection mechanism, BSM (C.N. Nanev, Progress in Crystal Growth and Characterization of Materials, 59 , 133–169, 2013) allows explaining a set of traits in both protein crystal nucleation and growth processes. BSM explanatory and predictive power are enhanced now, when intra‐crystalline repulsive interactions are assumed to act in parallel with the attractive forces, the former arising due to protein surface patch‐to‐patch incompatibility. Shapes of 1D and 2D protein crystals are considered from such a perspective. Using BSM the strong directional kinetic anisotropy in the edge growth rates of 2D protein crystals is tackled. The shapes of near‐critically sized apoferritin crystals and of experimentally grown 3D apoferritin crystals are considered.     

Structural Investigations of RNA–Protein Complexes in Post-Ribosomal Era

Crystallography Reports - Structural studies of RNA–protein complexes are important for understanding many molecular mechanisms occurring in cells (e.g., regulation of protein synthesis and...     

On slow protein crystal nucleation: cluster‐cluster aggregation on diffusional encounters

With a view to experimental results on protein crystal nucleation the effects of cluster coalescence are probed semi‐quantitatively. The steric association restriction, which stems from the patchy surface of the protein molecules, explains both experimentally measured low crystal nucleation rate and coalescence limitations for crystalline clusters of protein molecules. The conclusion is that due to its action, and the impact of rotational diffusion, the coalescence of critical (and/or supercritical) clusters should be rejected as a conceivable alternative for explaining the slow nucleation of protein crystals. Besides, the analysis of cluster‐cluster aggregation on diffusional encounters may be of more general interest; it may be helpful by considering the coalescence of structured bio‐nano‐particles. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)