Growing Crystals for X-ray Free-Electron Laser Structural Studies of Biomolecules and Their Complexes.

Currently, X-ray crystallography, which typically uses synchrotron sources, remains the dominant method for structural determination of proteins and other biomolecules. However, small protein crystals do not provide sufficiently high-resolution diffraction patterns and suffer radiation damage; therefore, conventional X-ray crystallography needs larger protein crystals. The burgeoning method of serial crystallography using X-ray free-electron lasers (XFELs) avoids these challenges: it affords excellent structural data from weakly diffracting objects, including tiny crystals. An XFEL is implemented by irradiating microjets of suspensions of microcrystals with very intense X-ray beams. However, while the method for creating microcrystalline microjets is well established, little attention is given to the growth of high-quality nano/microcrystals suitable for XFEL experiments. In this study, in order to assist the growth of such crystals, we calculate the mean crystal size and the time needed to grow crystals to the desired size in batch crystallization (the predominant method for preparing the required microcrystalline slurries); this time is reckoned theoretically both for microcrystals and for crystals larger than the upper limit of the Gibbs-Thomson effect. The impact of the omnipresent impurities on the growth of microcrystals is also considered quantitatively. Experiments, performed with the model protein lysozyme, support the theoretical predictions.

Protein Crystals Nucleated and Grown by Means of Porous Materials Display Improved X-ray Diffraction Quality.

Well-diffracting protein crystals are indispensable for X-ray diffraction analysis, which is still the most powerful method for structure-function studies of biomolecules. A promising approach to growing such crystals is the use of porous nucleation-inducing materials. However, while protein crystal nucleation in pores has been thoroughly considered, little attention has been paid to the subsequent growth of crystals. Although the nucleation stage is decisive, it is the subsequent growth of crystals outside the pore that determines their diffraction quality. The molecular-scale mechanism of growth of protein crystals in and outside pores is theoretically considered. Due to the low degree of metastability, the crystals that emerge from the pores grow slowly, which is a prerequisite for better diffraction. This expectation has been corroborated by experiments carried out with several types of porous material, such as bioglass ("Naomi's Nucleant"), buckypaper, porous gold and porous silicon. Protein crystals grown with the aid of bioglass and buckypaper yield significantly better diffraction quality compared with crystals grown conventionally. In all cases, visually superior crystals are usually obtained. Our theoretical conclusion is that heterogeneous nucleation of a crystal outside the pore is an exceptional case. Rather, the protein crystals nucleating inside the pores continue growing outside them.

Theoretical and experimental investigation of protein crystal nucleation in pores and crevices.

The nucleation ability of pores is explained using the equilibration between the cohesive energy maintaining the integrity of a crystalline cluster and the destructive energy tending to tear it up. It is shown that to get 3D crystals it is vital to have 2D crystals nucleating in the pores first. By filling the pore orifice, the 2D crystal nuclei are more stable because their peripheries are protected from the destructive action of water molecules. Furthermore, the periphery of the 2D crystal is additionally stabilized as a result of its cohesion with the pore wall. The understanding provided by this study combining theory and experiment will facilitate the design of new nucleants.

Hydrophobic Interface-Assisted Protein Crystallization: Theory and Experiment.

Macromolecular crystallization is crucial to a large number of scientific fields, including structural biology; drug design, formulation, and delivery; manufacture of biomaterials; and preparation of foodstuffs. The purpose of this study is to facilitate control of crystallization, by investigating hydrophobic interface-assisted protein crystallization both theoretically and experimentally. The application of hydrophobic liquids as nucleation promoters or suppressors has rarely been investigated, and provides an underused avenue to explore in protein crystallization. Theoretically, crystal nucleation is regarded as a two-step process, the first step being a local increase in protein concentration due to its adsorption on the hydrophobic surface. Subsequently, the protein is ordered in a crystal lattice. The energetic aspect of crystal nucleation on water/hydrophobic substance interfaces is approached by calculating the balance between the cohesive energy maintaining integrity of the two-dimensional crystal nucleus and the sum of destructive energies tending to tear up the crystal. This is achieved by comparing the number of bonds shared by the units forming the crystal and the number of unshared (dangling) bonds on the crystal surface pointing toward the solution. The same approach is extended to three-dimensional protein crystal nucleation at water/hydrophobic liquid interfaces. Experimentally, we studied protein crystallization over oils and other hydrophobic liquids (paraffin oil, FC-70 Fluorinert fluorinated oil, and three chlorinated hydrocarbons). Crystallizations of α-lactalbumin and lysozyme are compared, and additional information is acquired by studying α-crustacyanin, trypsin, an insulin analogue, and protein Lpg2936. Depending on the protein type, concentration, and the interface aging time, the proteins exhibit different crystallization propensities depending on the hydrophobic liquid used. Some hydrophobic liquids provoke an increase in the effective supersaturation, which translates to enhancement of crystal nucleation at their interface with the crystallization solution, leading to the formation of crystals.

Protein crystal nucleation in pores.

The most powerful method for protein structure determination is X-ray crystallography which relies on the availability of high quality crystals. Obtaining protein crystals is a major bottleneck, and inducing their nucleation is of crucial importance in this field. An effective method to form crystals is to introduce nucleation-inducing heterologous materials into the crystallization solution. Porous materials are exceptionally effective at inducing nucleation. It is shown here that a combined diffusion-adsorption effect can increase protein concentration inside pores, which enables crystal nucleation even under conditions where heterogeneous nucleation on flat surfaces is absent. Provided the pore is sufficiently narrow, protein molecules approach its walls and adsorb more frequently than they can escape. The decrease in the nucleation energy barrier is calculated, exhibiting its quantitative dependence on the confinement space and the energy of interaction with the pore walls. These results provide a detailed explanation of the effectiveness of porous materials for nucleation of protein crystals, and will be useful for optimal design of such materials.

Hypergravity as a crystallization tool.

The centrifugal increase of concentration is nondestructive, rapid, and simple technology. Therefore it is used to create a higher supersaturation that is required for crystal nucleation, as the one that is appropriate for the subsequent growth. Crystal nucleation is evoked in glass capillary tubes filled with protein solutions. The couple ferritin/ apoferritin is used as model proteins in the present article. Although differing in their masses the two (quasi) spherical molecules have exactly the same size and surface properties. Together with the temperature-independent solubility this makes them very convenient for our investigations. Decoupling nucleation and growth, for example, by means of hypergravity makes it possible to grow quasi-equidimensional crystals. The use of monodisperse crystalline forms of therapeutic agents can ensure constant time-release of protein-based medications.

Is crystal growth under low supersaturations influenced by a tendency to a minimum of the surface-free energy?

The long-standing problem of face morphology is discussed. Special emphasis is put on macroscopically flat faces, whose growth, under low supersaturations, is driven by dislocations possessing some screw component. The most general case, where the crystal face is pierced by more than one screw dislocation, is considered. If performed under sufficiently low supersaturation, the growth leads to the formation of the face morphology corresponding to the minimum of the surface-free energy. The thermodynamic driving force for face flattening is the difference in the surface-free energy of the vicinal faces of the hillocks (emanating from screw dislocations) and that of a singular face, which can truncate the valley between the growth hillocks. The hillock slope gives the quantitative relation between energetics and kinetics. The result of the considerations is that the lower the supersaturation, the more important is the role of the surface-free energy in face flattening. Another factor, particularly under sufficiently low supersaturations, is the effective increase in the local supersaturation at the valley separating two growth hillocks. The reason is that the dislocation strain energy vanishes there. (This is most evident when two dislocations of opposite sign are considered.) Besides, the valley floors are concave regions on the crystal surface, where the building blocks are bound more strongly. Thus, the kinetic reason for face flattening is the relative preference for incorporation of atoms, arriving from the ambient phase, at the valley floor. Note that accelerated step annihilation in the valley floor should be a universal factor, which favors face flattening under any supersaturation. The amount of flattening in the growth situation is determined by the interplay between supersaturation and thermodynamics.

Effects of buoyancy-driven convection on nucleation and growth of protein crystals.

Protein crystallization has been studied in presence or absence of buoyancy-driven convection. Gravity-driven flow was created, or suppressed, in protein solutions by means of vertically directed density gradients that were caused by generating suitable temperature gradients. The presence of enhanced mixing was demonstrated directly by experiments with crustacyanin, a blue-colored protein, and other materials. Combined with the vertical tube position the enhanced convection has two main effects. First, it reduces the number of nucleated hen-egg-white lysozyme (HEWL) crystals, as compared with those in a horizontal capillary. By enabling better nutrition from the protein in the solution, convection results in growth of fewer larger HEWL crystals. Second, we observe that due to convection, trypsin crystals grow faster. Suppression of convection, achieved by decreasing solution density upward in the capillary, can to some extent mimic conditions of growth in microgravity. Thus, impurity supply, which may have a detrimental effect on crystal quality, was avoided.

Nucleation of insulin crystals in a wide continuous supersaturation gradient.

Modifying the classical double pulse technique, by using a supersaturation gradient along an insulin solution contained in a glass capillary tube, we found conditions appropriate for the direct measurement of nucleation parameters. The nucleation time lag has been measured. Data for the number of crystal nuclei versus the nucleation time were obtained for this hormone. Insulin was chosen as a model protein because of the availability of solubility data in the literature. A comparison with the results for hen-egg-white lysozyme, HEWL was performed.

Nucleation rate determination by a concentration pulse technique: application on ferritin crystals to show the effect of surface treatment of a substrate.

The nucleation of horse spleen ferritin (HSF) crystals on substrates was investigated using a new modification of the double pulse technique. The influence of three different structureless substrates (glass, glass covered by methyl groups and poly-L-lysin template) on the nucleation was studied. The boundaries in the phase-diagram, which separate zones of crystal nucleation and growth were obtained by keeping pH = 5.0, and using CdSO(4) as crystallizing agent. The steady-state nucleation rates were determined. The energy required for critical nuclei formation was evaluated (10(-13) erg) and the sizes of critical nuclei were found (5 and 2 molecules).