Direct observation of nucleus structure and nucleation pathways in apoferritin crystallization.

Using atomic force microscopy (AFM) in situ during the crystallization of the protein apoferritin from its solution, we imaged the arrangement of the molecules in near-critical clusters, larger or smaller than the crystal nucleus, that are representative of the nucleus structure. At supersaturations Delta mu/k(B)T of 1.1 -- 1.6 -- 2.3, the nuclei contain about 50 -- 20 -- 10 molecules. The molecular arrangement within the nuclei is similar to that in the crystal bulk. Contrary to the general belief, the observed nuclei are not compact molecular clusters, but are planar arrays of several rods of 4--7 molecules set in one or two monomolecular layers. Similarly unexpected nuclei structures might be common, especially for anisotropic molecules. Hence, the nucleus structure should be considered as a variable by advanced theoretical treatments.

Molecular mechanisms of microheterogeneity-induced defect formation in ferritin crystallization.

We apply in situ atomic force microscopy to the crystallization of ferritins from solutions containing approximately 5% (w/w) of their inherent molecular dimers. Molecular resolution imaging shows that the dimers consist of two bound monomers. The constituent monomers are likely partially denatured, resulting in increased hydrophobicity of the dimer surface. Correspondingly, the dimers strongly adsorb on the crystal surface. The adsorbed dimers hinder step growth and on incorporation by the crystal initiate stacks of up to 10 triple and single vacancies in the subsequent crystal layers. The molecules around the vacancies are shifted by approximately 0.1 molecular dimensions from their crystallographic positions. The shifts strain the lattice and, as a consequence, at crystal sizes > 200 microm, the accumulated strain is resolved by a plastic deformation whereupon the crystal breaks into mosaic blocks 20-50 microm in size. The critical size for the onset of mosaicity is similar for ferritin and apoferritin and close to the value for a third protein, lysozyme; it also agrees with theoretical predictions. Trapped microcrystals in ferritin and apoferritin induce strain with a characteristic length scale equal to that of a single point defect, and, as a consequence, trapping does not contribute to the mosaicity. The sequence of undesired phenomena that include heterogeneity generation, adsorption, incorporation, and the resulting lattice strain and mosaicity in this and other proteins systems, could be avoided by improved methods to separate similar proteins species (microheterogeneity) or by increasing the biochemical stability of the macromolecules against oligomerization.

Differential pathways in oxy and deoxy HbC aggregation/crystallization.

CC individuals, homozygous for the expression of beta(C)-globin, and SC individuals expressing both beta(S) and beta(C)-globins, are known to form intraerythrocytic oxy hemoglobin tetragonal crystals with pathophysiologies specific to the phenotype. To date, the question remains as to why HbC forms in vivo crystals in the oxy state and not in the deoxy state. Our first approach is to study HbC crystallization in vitro, under non-physiological conditions. We present here a comparison of deoxy and oxy HbC crystal formation induced under conditions of concentrated phosphate buffer (2g% Hb, 1. 8M potassium phosphate buffer) and viewed by differential interference contrast microscopy. Oxy HbC formed isotropic amorphous aggregates with subsequent tetragonal crystal formation. Also observed, but less numerous, were twisted, macro-ribbons that appeared to evolve into crystals. Deoxy HbC also formed aggregates and twisted macro-ribbon forms similar to those seen in the oxy liganded state. However, in contrast to oxy HbC, deoxy HbC favored the formation of a greater morphologic variety of aggregates including polymeric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons. Unlike the oxy (R-state) tetragonal crystal, deoxy HbC formed flat, hexagonal crystals. These results suggest: (1) the Lys substitution at beta6 evokes a crystallization process dependent upon ligand state conformation [i. e., the R (oxy) or T (deoxy) allosteric conformation]; and (2) the oxy ligand state is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. This is in contrast to the deoxy form of HbC that energetically equally favors multiple pathways of aggregation, not all of which might culminate in crystal formation.

Molecular-level thermodynamic and kinetic parameters for the self-assembly of apoferritin molecules into crystals.

The self-assembly of apoferritin molecules into crystals is a suitable model for protein crystallization and aggregation; these processes underlie several biological and biomedical phenomena, as well as for protein and virus self-assembly. We use the atomic force microscope in situ, during the crystallization of apoferritin to visualize and quantify at the molecular level the processes responsible for crystal growth. To evaluate the governing thermodynamic parameters, we image the configuration of the incorporation sites, "kinks", on the surface of a growing crystal. We show that the kinks are due to thermal fluctuations of the molecules at the crystal-solution interface. This allows evaluation of the free energy of the intermolecular bond phi=3.0 k(B)T=7.3 kJ/mol. The crystallization free energy, extracted from the protein solubility, is -42 kJ/mol. Published determinations of the second virial coefficient and the protein solubility between 0 and 40 degrees C revealed that the enthalpy of crystallization is close to zero. Analyses based on these three values suggest that the main component in the crystallization driving force is the entropy gain of the water molecules bound to the protein molecules in solution and released upon crystallization. Furthermore, monitoring the incorporation of individual molecules in to the kinks, we determine the characteristic frequency of attachment of individual molecules at one set of conditions. This allows a correlation between the mesoscopic kinetic coefficient for growth and the molecular-level thermodynamic and kinetic parameters determined here. We found that step growth velocity, scaled by the molecular size, equals the product of the kink density and attachment frequency, i.e. the latter pair are the molecular-level parameters for self-assembly of the molecules into crystals.

Evidence for non-DLVO hydration interactions in solutions of the protein apoferritin.

We have studied molecular interactions in solutions of the protein apoferritin by static and dynamic light scattering. When plotted against the electrolyte concentration, the second osmotic virial coefficient exhibits a minimum. The ascending branch of this dependence is a manifestation of a surprisingly strong repulsion between the molecules at electrolyte concentrations about and above 0.2M, where electrostatic interactions are suppressed. We argue that the repulsion is due to the water structuring, enhanced by the accumulation of hydrophilic counterions around the apoferritin molecules, giving rise to so-called hydration forces.

Quasi-planar nucleus structure in apoferritin crystallization.

First-order phase transitions of matter, such as condensation and crystallization, proceed through the formation and subsequent growth of 'critical nuclei' of the new phase. The thermodynamics and kinetics of the formation of these critical nuclei depend on their structure, which is often assumed to be a compact, three-dimensional arrangement of the constituent molecules or atoms. Recent molecular dynamics simulations have predicted compact nucleus structures for matter made up of building blocks with a spherical interaction field, whereas strongly anisotropic, dipolar molecules may form nuclei consisting of single chains of molecules. Here we show, using direct atomic force microscopy observations, that the near-critical-size clusters formed during the crystallization of apoferritin, a quasi-spherical protein, and which are representative of the critical nucleus of this system, consist of planar arrays of one or two monomolecular layers that contain 5-10 rods of up to 7 molecules each. We find that these clusters contain between 20 and 50 molecules each, and that the arrangement of the constituent molecules is identical to that found in apoferritin crystals. We anticipate that similarly unexpected critical nucleus structures may be quite common, particularly with anisotropic molecules, suggesting that advanced nucleation theories should treat the critical nucleus structure as a variable.

Control of protein crystal nucleation around the metastable liquid-liquid phase boundary.

The capability to enhance or suppress the nucleation of protein crystals opens opportunities in various fundamental and applied areas, including protein crystallography, production of protein crystalline pharmaceuticals, protein separation, and treatment of protein condensation diseases. Herein, we show that the rate of homogeneous nucleation of lysozyme crystals passes through a maximum in the vicinity of the liquid-liquid phase boundary hidden below the liquidus (solubility) line in the phase diagram of the protein solution. We found that glycerol and polyethylene glycol (which do not specifically bind to proteins) shift this phase boundary and significantly suppress or enhance the crystal nucleation rates, although no simple correlation exists between the action of polyethylene glycol on the phase diagram and the nucleation kinetics. The control mechanism does not require changes in the protein concentration, acidity, and ionicity of the solution. The effects of the two additives on the phase diagram strongly depend on their concentration, which provides opportunities for further tuning of nucleation rates.

Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes.

We have studied 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 Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although 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 applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. 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 and other phase transitions in the protein solution.

Intrinsic instability of the concentration field in diffusion-limited growth and its effect on crystallization.

The dynamic behavior of the concentration field in crystallization is investigated by considering the coupling of the bulk concentration field and interfacial kinetics. It is shown that the concentration field may become unstable for perturbations with certain wavelength. When instability occurs, the physical environment in front of the growing interface will fluctuate and the interfacial growth mode will be affected accordingly. We suggest that our analysis can be used to interpret some spatial-temporal instabilities observed in crystallization.

Protein crystal growth--microgravity aspects.

Protein crystals, grown under reduced gravity conditions, are either superior or inferior in their structural perfection than their Earth-grown counterparts. A reduction of the crystals' quality due to low-gravity effects on the growth processes cannot be understood from existing models. In this paper we put forth a rationale which predicts either advantages or disadvantages of microgravity growth. This rationale is based on the changes in the effective solute and impurity supply rates in microgravity and their effects on the intrinsic growth rate fluctuations that arise from the coupling of bulk transport to nonlinear interfacial kinetics and cause severe inhomogeneities. Depending on the specific diffusivity and kinetic coefficient of a protein and the impurities in the solution, either transport enhancement through forced flow or transport suppression under reduced gravity can result in a reduction of the kinetic fluctuations and, thus, growth with higher structural perfection. Investigating this mechanism of microgravity effects, we first demonstrate a one-to-one correspondence between these fluctuations, that are due to the bunching of growth steps, and the formation of defects in the crystals. We have confirmed the forced flow aspects of this rationale in ground-based experiments with lysozyme utilizing flowing solutions with varying, well characterized impurity contents.

Effects of microheterogeneity in hen egg-white lysozyme crystallization.

In earlier sodium dodecylsulfate-polyacylamide gel electrophoresis (SDS-PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026-0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS-PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS-PAGE analysis of OHEWL yielded two closely spaced dimer bands with Mr = 28000 and 27500. In addition, larger HEWL oligomers with Mr = 1.7 million and 320000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 microgram-1 mg ml-1 OHEWL to 32 mg ml-1 HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03-0.3 M hydrogen peroxide. Repartitioning of the dimer during crystallization at various temperatures between 277 and 293 K was analyzed by SDS-PAGE. The crystals contained

Heterogeneity determination and purification of commercial hen egg-white lysozyme.

Hen egg-white lysozyme (HEWL) is widely used as a model protein, although its purity has not been adequately characterized by modern biochemical techniques. We have identified and quantified the protein heterogeneities in three commercial HEWL preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis with enhanced silver staining, reversed-phase fast protein liquid chromatography (FPLC) and immunoblotting with comparison to authentic protein standards. Depending on the source, the contaminating proteins totalled 1-6%(w/w) and consisted of ovotransferrin, ovalbumin, HEWL dimers, and polypeptides with approximate M(r) of 39 and 18 kDa. Furthermore, we have obtained gram quantities of electrophoretically homogeneous [> 99.9%(w/w)] HEWL by single-step semi-preparative scale cation-exchange FPLC with a yield of about 50%. Parallel studies of crystal growth kinetics, salt repartitioning and crystal perfection with this highly purified material showed fourfold increases in the growth-step velocities and significant enhancement in the structural homogeneity of HEWL crystals.

Repartitioning of NaCl and protein impurities in lysozyme crystallization.

Nonuniform precipitant and impurity incorporation in protein crystals can cause lattice strain and, thus, possibly decrease the X-ray diffraction resolution. To address this issue, a series of crystallization experiments were carried out, in which initial supersaturation, NaCl concentration, protein purity level and crystallized fraction were varied. Lysozyme and protein impurities, as well as sodium and chloride were independently determined in the initial solution, supernatant and crystals. The segregation coefficients for Na(+) and Cl(-) were found to be independent of supersaturation and NaCl concentration, and decreased with crystallized fraction/crystal size. Numerical evaluation of the extensive body of data, based on a nucleation-growth-repartitioning model, suggests a core of approximately 40 micro m in which salt is incorporated in much greater concentrations than during later growth. Small crystals containing higher amounts of incorporated NaCl also had higher protein impurity contents. This suggests that the excess salt is associated with the protein impurities in the core. X-ray topography revealed strain fields in the center of the crystals comparable in size to the inferred core. The growth rates of crystals smaller than 30-40 micro m in size were consistently 1.5-2 times lower than those of larger crystals, presumably due to higher chemical potentials in the core.

Growth process of protein crystals revealed by laser Michelson interferometry investigation.

The elementary processes of protein crystal growth were investigated by means of laser Michelson interferometry on the example of the (101) face of tetragonal hen egg-white (HEW) lysozyme. The method allows real-time in situ observations of the morphology of the growing protein crystal surface, as well as simultaneous precise measurement of growth rate and step velocity on identified growth-layer sources. At the critical supersaturation of 1.6 the growth mechanism was shown to transform from dislocation-layer generation to surface nucleation. Measurements on different growth hillocks, with material of a different source and at a different temperature, all indicated that for supersaturations lower than approximately 1 growth is hindered by the competitive adsorption of (most probably) other protein species contained in HEW, although the material is pure by most analytical methods. At supersaturations sigma < 0.4 other impurities sometimes led to cessation of growth. However, at sigma in the range 0.9 < sigma < 1.6 growth processes are determined by the kinetics of pure lysozyme. This enabled us to measure the step kinetic coefficient beta for crystallization of a protein substance for the first time: beta = 2.8 micro m s(-1). This also means that by working in this supersaturation range we can eliminate the impurity effects. Other means to reduce influence of impurities is to use, if possible, a higher crystallization temperature. It is shown that slow crystallization of proteins is due primarily to impedance of the elementary act of entering the growth site and not to the low concentration of the solution. The value of beta does not depend on temperature, indicating the decisive role of entropy, not energy barriers, in the crystallization of biological macromolecules.