Christo N. Nanev

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.

Heterogeneous nucleation (and adhesion) of lysozyme crystals

Abstract The heterogeneous nucleation of hen-egg-white lysozyme (HEWL) crystals has been investigated using a double-(thermal)-pulse technique, thus detaching nucleation from the growth stage. n ( t ) dependencies of the nucleus number, n , vs. time, t , were plotted for surfaces rendered hydrophobic (by means of hexamethyl-disilazane) and for bare glass surfaces. Preferred crystallite orientation supplied additional information. The discussion is based on the classical Stranski–Kaischew theory of crystal nucleation. With a purposely developed new technique the adhesion energies have been measured for HEWL crystals, grown on different supports.

Temperature-independent solubility and interactions between apoferritin monomers and dimers in solution

Abstract We used chromatographic, static and dynamic light scattering techniques, and atomic force microscopy (AFM) to study the structure of the protein species and the protein–protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of NaAc buffer and CdSO 4 . The sizes and shapes of the monomers and dimers, separated by size-exclusion chromatography, were determined by dynamic light scattering and AFM. While the monomer is an apparent sphere with a diameter corresponding to previous X-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was used to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The addition of even small amounts of Cd 2+ causes attraction between the monomer molecules. Furthermore, we found that the second virial coefficient and the protein solubility do not noticeably depend on temperature in the range from 0°C to 40°C. This suggests that the enthalpy for crystallization of apoferritin is close to zero, and the gain of entropy is essentially constant in this temperature range. We also found that in solutions of the apoferritin dimer, the molecules attract even in the presence of acetate buffer only, indicating a change in the surface of the apoferritin molecule. In view of the repulsion between the monomers at the same conditions, this suggests that the dimers and higher oligomers form only after partial unfolding of some of the apoferritin subunits. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization in the protein solution.

Heterogeneous Nucleation of Hen-Egg-White Lysozyme — Molecular Approach

The heterogeneous nucleation of hen-egg-white lysozyme (HEWL) crystals has been repeatedly investigated using a double-(thermal)-pulse technique, thus detaching nucleation from growth stage. n(t) dependencies of the nucleus number n, on templates of poly-L-lysine, vs time, t were plotted and the steady-state nucleation rates I were determined. They were compared with the results obtained earlier for surfaces rendered hydrophobic (by means of hexamethyl-disilazane) as well as for bare glass surfaces. In the present paper we determine the number of HEWL molecules in the (heterogeneously formed) critical nucleus. It turned out that it is build of 3 (to 4) HEWL molecules on glass substrate and 8 molecules for both hexamethyl-disilazane and poly-L-lysine templates. The energy A k required for heterogeneous formation of a critical nucleus on poly-L-lysine has been calculated, on the basis of the steady-state nucleation rates I. Intermolecular binding energy in the HEWL crystal lattice has been estimated again (approximately 10 -9 erg/molecule). This time the basis was the adhesion of HEWL crystals to poly-L-lysine substrate (compare D. TZEKOVA, S. DIMITROVA, C.N. NANEV, J. Cryst. Growth, 196 (2), 226 (1999)).

Polyhedral instability — skeletal and dendritic growth

Abstract The stability of flat facets is a fundamental problem in crystal growth. Simultaneously, it is also a prerequisite for producing high-quality homogeneous crystals, so essential for the high-tech applications. The preservation of the crystal shape during crystal growth, however, is a complex problem It involves a specific interplay between: mass/and heat transfer to/from the crystal surface, and the kinetics of the elementary incorporation, i.e. between the two major successive steps in the growth process. Rapid growth, and hence diffusion mechanism limitations, can result in various imperfections; such as solvent inclusions, the capture of impurities, the incorporation of veils and the formation of cavities in the facets. Skeletal and dendritic patterns are the following stages. The importance of these crystal forms can be illustrated with ice and snow crystals, and in connection with their role in the nature. The review includes also discussion of skeletal crystal shapes, very familiar to the snow-flakes and dendrites of ice. The problem for the polyhedral instability concerns also the mineralogy, etc.

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).

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.

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.

Instability of Faceted Crystal Shapes and their Transformation into Skeletons during Growth under Diffusion Control

Abstract The (in-)stability of polyhedral forms of crystals which grow under conditions of hindered transport in the parent phase is reviewed. Together with classical results, new ideas on the explanation of the process have been considered. Experimental results on the growth of crystals from the vapour phase, from aqueous solutions and during electrocrystallization are the basis for treatment. The parameters causing the loss of stability, and the transition from isometric to skeletal and dendritic growth of crystals elucidated experimentally, have been compared with the predictions of a phenomenological theory of polyhedral stability, which is based on Seegers approximation for the inhomogeneous concentration distribution about the crystal face growing under diffusion limitation. Intensive efforts have been made to investigate the details of the step kinetics leading to the appearance of shallow cavities in the crystal faces. At the end, for comparison purposes, the growth of some peculiar crystal forms...

Theory of Nucleation

Abstract The principles of the classical nucleation theory (CNT), originating from J.W. Gibbs, are presented in this chapter. Then, thermodynamics, critical nucleus size, energy barriers, and stationary and nonstationary nucleation kinetics are considered as the basis for further theoretical developments; attention is devoted to nucleation induction time. Gas bubble formation and crystal nucleation in vapors, liquids, melts, and solutions are discussed. Numerous attempts to correct CNT, as well as the probabilistic character of the nucleation and the so-called nucleation theorem, are mentioned. Also presented is the fruitful molecular-kinetic elaboration of CNT by Stranski and Kaischew. Its advantages are used for considering homogeneous, heterogeneous, and two-dimensional nucleation. Equilibrium crystal shapes, energy barriers, and the kinetics of protein crystal nucleation are treated from the same molecular-kinetic standpoint by introducing a concept for bond selection mechanism.