Apoferritin Crystals Research Articles

Solvent flows, conformation changes and lattice reordering in a cold protein crystal.

When protein crystals are abruptly cooled, the unit-cell, protein and solvent-cavity volumes all contract, but the volume of bulk-like internal solvent may expand. Outflow of this solvent from the unit cell and its accumulation in defective interior crystal regions has been suggested as one cause of the large increase in crystal mosaicity on cooling. It is shown that when apoferritin crystals are abruptly cooled to temperatures between 220 and 260 K, the unit cell contracts, solvent is pushed out and the mosaicity grows. On temperature-dependent timescales of 10 to 200 s, the unit-cell and solvent-cavity volume then expand, solvent flows back in, and the mosaicity and B factor both drop. Expansion and reordering at fixed low temperature are associated with small-amplitude but large-scale changes in the conformation and packing of apoferritin. These results demonstrate that increases in mosaicity on cooling arise due to solvent flows out of or into the unit cell and to incomplete, arrested relaxation of protein conformation. They indicate a critical role for time in variable-temperature crystallographic studies, and the feasibility of probing interactions and cooperative conformational changes that underlie cold denaturation in the presence of liquid solvent at temperatures down to ∼200 K.

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.

Layered crystals of apo‐ and holoferritin grown by alternating crystallization

AbstractWe 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)

Dependence of Apoferritin Crystal Growth on Temperature and Cadmium Concentration

Static light scattering and multiwell microbatch crystallization experiments have been used to determine how apoferritin crystal growth kinetics and final crystal size depend on temperature, pH, and cadmium concentration. The experiments were conducted on a linear thermal gradient covering a range from 30 to 40 °C. Crystal growth was monitored in situ under a microscope without disturbing the thermal environment. A dependence of apoferritin crystal formation and growth on temperature and cadmium concentration was observed. The range of cadmium concentrations that resulted in apoferritin crystal growth was evaluated and found to be consistent with the osmotic second virial coefficients determined via static light scattering. For the domain of conditions where apoferritin crystal formation occurred, the initial molar growth rate of apoferritin crystals increased linearly with temperature. It was observed that increasing cadmium concentration decreased the dependence of the final crystal size on temperature. The measured dependence of growth kinetics on temperature allowed the estimation of an activation energy for crystal growth. By comparing the value of this activation energy with that for nucleation (obtained from the literature), it became possible to explain the observation of larger, but fewer, apoferritin crystals at higher temperatures, which has also been reported in the literature for other proteins.

Experimental determination of the radiation dose limit for cryocooled protein crystals

Radiation damage to cryocooled protein crystals during x-ray structure determination has become an inherent part of macromolecular diffraction data collection at third-generation synchrotrons. Generally, radiation damage is an undesirable component of the experiment and can result in erroneous structural detail in the final model. The characterization of radiation damage thus has become an important area for structural biologists. The calculated dose limit of 2 x 10(7) Gy for the diffracting power of cryocooled protein crystals to drop by half has been experimentally evaluated at a third-generation synchrotron source. Successive data sets were collected from four holoferritin and three apoferritin crystals. The absorbed dose for each crystal was calculated by using the program raddose after measurement of the incident photon flux and determination of the elemental crystal composition by micro-particle-induced x-ray emission. Degradation in diffraction quality and specific structural changes induced by synchrotron radiation then could be compared directly with absorbed dose for different dose/dose rate regimes: a 10% lifetime decrease for a 10-fold dose rate increase was observed. Remarkable agreement both between different crystals of the same type and between apoferritin and holoferritin was observed for the dose required to reduce the diffracted intensity by half (D(1/2)). From these measurements, a dose limit of D(1/2) = 4.3 (+/-0.3) x10(7) Gy was obtained. However, by considering other data quality indicators, an intensity reduction to I(ln2) = ln2 x I(0), corresponding to an absorbed dose of 3.0 x 10(7) Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments.

The effects of temperature and radiation on holo and apo ferritin crystals

The effects of temperature and radiation on holo and apo ferritin crystals

Structural description of the active sites of mouse L-chain ferritin at 1.2 A resolution.

The first ferritin structure refined at the atomic level has been achieved on recombinant mouse L-chain apoferritin (rMoLF) crystals. These latter diffract to 1.2 A resolution under cryogenic conditions. When cryo-cooling the sample, the thermal disorder usually observed at room temperature is reduced and the low-temperature structure reveals several details concerning the protein putative active sites and their properties. Within the pores built up by the molecular three-fold symmetry axes, the iron entry route to the ferritin cavity, residues H118, D131 and E134, exhibit alternate conformations associated with the binding of partially hydrated cadmium ions, a metal used as a crystallization agent. At the mineral ferrihydrite nucleation center, the electron density maps evidence the orientation of E57, E60, E61 and E64 glutamate side chains (whereas they were observed highly disordered in previous ferritin structures determined at room temperature) and allow a description of the site taking into account the binding geometry of four Cd(2+) ions. Moreover, the side chain of residue K140, lying in the vicinity of the ferrihydrite nucleation center, is shown to interact with residue E61. As previously highlighted, this observation confirms the importance of K140 in the rMoLF sequence, as being responsible for the low level of iron incorporation by mousel L-chain ferritin compared to human L-chain ferritin. Finally, the diffusion of small molecules within the ferritin cavity is illustrated here by the presence of ordered molecules of glycerol used as a cryo-protectant, which bind the inner cavity surface of the protein.

Self-assembly of apoferritin molecules into crystals: thermodynamics and kinetics of molecular level processes

Aside from the biological, biomedical and materials importance of ferritin and apoferritin, the self-assembly of these molecules into crystals is of interest because it is a suitable model for protein crystallization and aggregation that impact many fundamental and applied fields. 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. 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 φ = 3.0 k B T = 7.3 kJ/mol. The crystallization free energy, extracted from the protein solubility, is − 42 kJ/mol. Thermodynamics analyses based on these two values suggest that the main component in the crystallization driving force is the entropy gain of the waters bound to the protein molecules in solution and released upon crystallization. Furthermore, we determine the characteristic frequency of attachment of individual molecules into the kinks at one set of conditions. We show that the macroscopic step growth velocity, scaled by the molecular size, equals the product of independently determined kink density and attachment frequency, i.e., these are the molecular-level parameters for crystallization. Finally, we show that although new crystal layers are generated by intrinsically stochastic surface nucleation, for crystals > 200 μm layer generation predominantly occurs at the crystal edges. Numerical simulations of the concentration fields in the solution allow us to assign this localization to higher interfacial concentration at the edges. As the steps propagate to cover the crystal face, step density waves, or bunches, develop and may lead to non-uniformity and lower quality and utility of the growing crystal.

Visualization of the impurity depletion zone surrounding apoferritin crystals growing in gel with holoferritin dimer impurity

Colorless apoferritin ( M≅450 kDa) crystals were grown in the presence of red colored holoferritin dimers ( M≅900 kDa) within agarose gel by counterdiffusion with Cd 2+ as precipitant. In agreement with our previous quantitative measurements showing preferential trapping of dimers (segregation coefficient K=4), the apoferritin crystals became darkly colored while the gel solution around them was nearly colorless. This segregation coefficient and the absence of convective mixing resulted in a large colorless area surrounding the crystals which allowed us to demonstrate the existence of the impurity depletion zone by direct visual observation.

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

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.

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 φ=3.0 kBT=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°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.

Direct Visualization of an Impurity Depletion Zone

When a crystal incorporates more impurity per unit of its volume than the impurity concentration in solution, the solution in vicinity of the growing crystal is depleted with respect to the impurity I,2. With a stagnant solution, e. g. in microgravity or gels, an impurity depletion zone expands as the crystal grows and results in greater purity in most of the outer portion of the crystal than in the core. Crystallization in gel provides an opportunity to mimic microgravity conditions and visualize the impurity depletion zone. Colorless, transparent apoferritin (M congruent to 450 KDa) crystals were grown in the presence of red holoferritin dimer as a microheterogeneous impurity (M congruent to 900 KDa) within agarose gel by counterdiffusion with Cd(2+) precipitant. Preferential trapping of dimers, (distribution coefficient K = 4 (exp 1,2)) results in weaker red color around the crystals grown in the left tube in the figure as compared to the control middle tube without crystals. The left and the middle tubes contain colored ferritin dimers, the right tube contains colored trimers. The meniscus in the left tube separate gel (below) and liquid solution containing Cd(2+) (above). Similar solutions, though without precipitants, were present on top of the middle and right tube allowing diffusion of dimers and trimers. The area of weaker color intensity around crystals directly demonstrates overlapped impurity depletion zones.

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.

Crystallization and preliminary X-ray diffraction data of mouse L-chain apoferritin crystals.

Crystals of recombinant mouse L-chain apoferritin were obtained by the hanging-drop technique using ammonium sulfate as precipitant. Two crystal forms were observed in the same drop. The crystals belong to either the P2 monoclinic or to the P42(1)2 tetragonal space group. The monoclinic crystals diffracted to beyond 2.4 A resolution but were systematically twinned, while the tetragonal crystals diffracted to beyond 2.9 A. These crystallization conditions in the absence of metal salts should facilitate the study of the interaction between L-chain ferritins and heavy metals, particularly the iron core.

Simplex optimization of protein crystallization conditions

Simplex algorithms have been used to optimize for size, number and morphology of lysozyme and apoferritin crystals. This approach requires fewer experiments than the single-factor-at-a-time method or factorial designs and will be useful in conserving materials on the International Space Station. The simplex method has the possible advantage that it conserves on materials by reducing the number of experiments required to optimize a crystallization system. The process is iterative and exploratory and should allow optimum microgravity conditions to be determined which might very well be different from the optimum conditions on Earth. Because the simplex method uses simple mathematical operations to calculate the next set of crystallization conditions it will be easier for crystal growers to implement than factorial designs. Factorial experiments are based on varying all factors simultaneously at a limited number of factor levels. This results in a model that is used to determine the influence of each factor and their interactions. Factorial design experiments are especially useful at the beginning of an experimental study and as a screening tool to investigate a large number of factors. The simplex method is an optimization method which is model-independent and requires no fitting of models to data. Also, when applied to protein crystal growth the simplex method does not rely on an absolute quality score. Instead, with each iteration a comparison is made to the last experiment and the results are assigned as being “better or worse”. In this study, commercially obtained apoferritin was purified from 65% monomeric apoferritin to 92% monomeric apoferritin by size exclusion chromatography. Simplex optimization found the best apoferritin crystals were obtained at 15 mg/ml apoferritin, 2.0% CdSO 4, 25°C using the hanging drop vapor diffusion method of crystallization and at 24 mg/ml apoferritin, 1.5% CdSO 4, 25°C using the containerless crystallization method. For lysozyme, the simplex method found the best crystals at 19 mg/ml lysozyme, 7.0% (w/v) NaCl, pH 4.0, 25°C using the hanging drop vapor diffusion method of crystallization. For both proteins, the optimum conditions were found with less than ten experiments using very little protein. Finally, we report that the factors to be considered in the successful application of this method to crystallization are the number of variables to be studied, the initial conditions, step size and analysis of crystal quality.

Light scattering investigations of protein and virus crystal growth: ferritin, apoferritin and satellite tobacco mosaic virus

Quasi-elastic light scattering was used to investigate the nucleation and crystallization of apoferritin, ferritin and satellite tobacco mosaic virus (STMV). Supersaturation dependent critical nuclear size supportive of stable macromolecular crystal growth was estimated to be in the range of 5 to 65 monomer protein and virus particles. The interfacial free energy and the activation energy for apoferritin crystal growth was estimated to be 2.7 x 10-2 erg/cm2 and 8.5 kcal/mol, and for STMV 1.8 x 10-2 erg/cm2 and 10 kcal/mol, respectively. The data suggests that the rate limiting factor in STMV crystal growth is the diffusion of virus clusters to the growing crystal nucleus, while apoferritin crystallization is limited by surface kinetics. The aggregation rate is substantially higher for crystallization with a power law exponent of n = 0.33-0.54 (STMV) and n = 0.8-1.0 (apoferritin), while that associated with eventual amorphous precipitate was n = 0.20.

The structure of apoferritin: Molecular size, shape and symmetry from X-ray data

Apoferritin is the protein component of the iron-storage protein ferritin, and forms a shell round the iron oxide core. Iron-free apoferritin crystallizes in a cubic form isomorphous with one form of ferritin and having four molecules in a unit cell of side 184 A and apparent space group F 432. From the X-ray data apoferritin molecules have a molecular weight of 480,000 and a form approximating on the average at a resolution of 26 A to a spherical shell having an external radius of 61 ± 3 A and internal to external radius ratio about 0·6. The shell is constructed from subunits and chemical results suggest that there are about twenty of these. Recent X-ray photographs of wet apoferritin crystals are found to contain both sharp reflections, extending to spacings of 1/1·4 A −1 , and diffuse streaks indicative of a structure having “ordered disorder”. The X-ray patterns are interpreted as being due to molecules which are situated regularly on a face-centred lattice, but having different orientations at random, so that the apparent point-group 4:3:2† is produced statistically from molecules with lower symmetry. The diffraction patterns suggest that the molecules have pseudo-icosahedral symmetry, but not exact icosahedral nor tetrahedral symmetry. The molecules probably consist of twenty subunits situated at the vertices of a pentagonal dodecahedron with point group 5:2, 2:2:2 or 2. An examination of the diffraction pattern of orthorhombic ferritin shows that the symmetry is almost certainly 5:2.