Anita Penkova

Nucleation of lysozyme crystals under external electric and ultrasonic fields

Preferred orientation along c-axis of hen-egg-white lysozyme (HEWL) crystals has been observed in an external electric field. Besides, the HEWL crystals grew predominantly on the cathode side of the glass cell. These facts were explained on the basis of a concept for specific spatial distribution of the positive electric charges on the individual HEWL molecules, and thus attributed to the (preferred) orientation of individual HEWL molecules in the solution, under these conditions. Ultrasonic field redoubles the nucleation rate of HEWL crystals, but does not change the number of building units in the critical nucleus. Taking into account the intermolecular binding energy, we conclude that ultrasonic field accelerates nucleation due to breaking of the protein crystals.

Nucleation of Protein Crystals under the Influence of Solution Shear Flow

Abstract:  Several recent theories and simulations have predicted that shear flow could enhance, or, conversely, suppress the nucleation of crystals from solution. Such modulations would offer a pathway for nucleation control and provide a novel explanation for numerous mysteries in nucleation research. For experimental tests of the effects of shear flow on protein crystal nucleation, we found that if a protein solution droplet of ∼ 5 μL (2–3 mm diameter at base) is held on a hydrophobic substrate in an enclosed environment and in a quasi‐uniform constant electric field of 2 to 6 kV cm−1, a rotational flow with a maximum rate at the droplet top of ∼ 10 μm s−1 is induced. The shear rate varies from 10−3 to 10−1 s−1. The likely mechanism of the rotational flow involves adsorption of the protein and amphiphylic buffer molecules on the air–water interface and their redistribution in the electric field, leading to nonuniform surface tension of the droplet and surface tension‐driven flow. Observations of the number of nucleated crystals in 24‐ and 72‐h experiments with the proteins ferritin, apoferritin, and lysozyme revealed that the crystals are typically nucleated at a certain radius of the droplet, that is, at a preferred shear rate. Variations of the rotational flow velocity resulted in suppression or enhancement of the total number of nucleated crystals of ferritin and apoferritin, while all solution flow rates were found to enhance lysozyme crystal nucleation. These observations show that shear flow may strongly affect nucleation, and that for some systems, an optimal flow velocity, leading to fastest nucleation, exists. Comparison with the predictions of theories and simulations suggest that the formation of ordered nuclei in a “normal” protein solution cannot be affected by such low shear rates. We conclude that the flow acts by helping or suppressing the formation of ordered nuclei within mesoscopic metastable dense liquid clusters. Such clusters were recently shown to exist in protein solutions and to constitute the first step in the nucleation mechanism of many protein and nonprotein systems.

Nucleation and growth of lysozyme crystals under external electric field

Preferred orientation along c-axis of hen-egg-white lysozyme (HEWL) crystals has been observed in an external electric field. This fact was explained on the basis of a working hypothesis for specific spatial distribution of the positive electric charges on the individual HEWL molecules, and thus attributed to the (preferred) orientation of individual HEWL molecules in the solution, under these conditions. Besides, the HEWL crystals grew predominantly on the cathode side of the glass cell. Due to the electric field the crystals grew faster. These observations have been explained on the basis of recently reported measurements of Aubry and co-workers, which show an increased HEWL concentration in the solution near the cathode, and lower one near the anode.

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

Abstract: 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.

Abstract: 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.

Polyhedral (in-)stability of protein crystals

Abstract The polyhedral (in-)stability of monoclinic hen-egg white lysozyme (HEWL) crystals, grown by means of PEG-6000, and that of orthorhombic trypsin crystals has been investigated experimentally. On the basis of a quantitative theoretical analysis, it is compared with the polyhedral (in-)stability of tetragonal HEWL and cubic ferritin crystals. The unambiguous conclusion is that the phenomenon is due to the diffusive supply of matter. This conclusion is also supported by the fact that the phenomenon has common features for both proteins and small molecular crystals.

Crystal Structure of Poly[(acetone-O)-3-((3,4-dimethoxyphenyl)(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-(2-oxo-2H-chromen-4-olate)sodium]

The structure of Poly[(acetone-O)-3-((3,4-dimethoxyphenyl)(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-(2-oxo-2H-chromen-4-olate)sodium] was determined by X-ray crystallography. The compound crystallizes in an orthorhombic system and was characterized thus P   , (2) A, (3) A, A. , (10) A3. The crystal structure was solved by direct methods and refined by full-matrix least-squares on to final values of and .

Instability of protein drops via applied electric field: mathematical and experimental aspects.

Drops (5–15 μL) consisting of a protein solution readily crystallize and could provide an opportunity for a simultaneous examination of their thermodynamic and kinetic properties at various sizes. These drops experienced different pressures and therefore different surface tensions. Starting from the expression for the interface traction between protein fluid and silicon medium (with different dielectric constants), we have derived an equation accounting the influence of the electric field strength on the geometry of a protein drop. If the field strength increases, the lysozyme drop between two electrodes elongates and some crystals nucleate on the cathode side. In this situation numerous factors besides the intensity of the electric field—such as the solution composition, the charge and size of the protein molecule, the purity of the protein substance, and the consistency of bubbles of water—can have a significant effect on the crystallization rate and location.