Two types of amorphous protein particles facilitate crystal nucleation (original) (raw)
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Heterogeneous versus bulk nucleation of lysozyme crystals
Crystal Research and Technology, 2010
Heterogeneous (on-glass) protein crystal nucleation was separated from the bulk one in systems of thin protein solution layers, confined between two glass plates of custom made quasi two-dimensional all-glass cells, as well as by applying forced protein solution flow. Two commercial samples of hen-egg-white lysozyme, Seikagaku and Sigma were used as model proteins. Applying the classical technique of separation in time of nucleation and growth stages with protein solution layers of thickness 0.05 cm we found that the on-glass crystal nucleation prevailed highly with Seikagaku HEWL, while on the opposite, bulk nucleated crystals represented the main crystal fraction in Sigma solution. Also using 0.05 cm solution layers nucleation rates were measured separately for the on-glass and bulk protein crystals. The process was investigated by varying solution layer thicknesses as well, from 0.05 down to 0.01, 0.0065 and 0.002 cm. Studying the influence of the forced protein solution flow on HEWL crystal nucleation the classical double-pulse technique was modified by separating the nucleation and growth stages not only in time, but simultaneously also in place. In this case we found that the ratio of on-glass formed crystal nuclei to bulk nuclei depended on the flow velocity, but in different manner with Seikagaku HEWL and Sigma HEWL. A plausible explanation of our experimental results is that the bulk crystal nucleation occurs on foreign surfaces as well, e.g. on rests of source biomaterial, which are always present in the protein solutions. Moreover, biomaterial seems to be more active nucleant than glass.
Recent advances in the understanding of two-step nucleation of protein crystals
Faraday Discussions, 2015
The two-step mechanism of nucleation of crystals in solutions posits that the formation of crystal nuclei occurs within structures of extended lifetimes, in which the nucleating solute is at high concentration. The validity of this mechanism has been demonstrated for proteins, small-molecule organic and inorganic materials, colloids, and polymers. Due to large molecule sizes, proteins are an ideal system to study the details of this nucleation pathway, in particular the formation mechanisms of the nucleation precursors and the associated physico-chemical rules. The precursors of protein crystal nuclei are protein-rich clusters of sizes ∼100 nm that contain 10 000–100 000 molecules and occupy less than 10−3of the total solution volume. Here we demonstrate, using oblique illumination microscopy, the liquid nature of the clusters of the protein lysozyme and reveal their inhomogeneous structure. We test a hypothesis put forth by theory that clusters primarily consist of transient protei...
Nucleation and Crystallization of Lysozyme: Role of Substrate Surface Chemistry and Topography
In an effort to better understand, direct and control the crystallization of molecular and macromolecular compounds, an approach using colloidal templates as substrates for heterogeneous nucleation was investigated. These templates combine both tuneable chemical functionalities and geometrical features, altering the crystal-substrate interactions. Colloidal templates were prepared from silica nanoparticles, where the surface chemistry was modified by silanization. Particle size varied from 30 to 700 nm, with silanols, NH 2 , CF 3 , phenyl, or dodecyl as surface functional groups. Here, we report on the template assisted crystallization of chicken egg white lysozyme (CEWL). Nucleation was dramatically affected by the surface chemistry and topography of the templates. Using 220 nm particles, hydrophobic templates generally produced fewer, larger crystals, while a larger number of small crystals were obtained on hydrophilic templates. The use of different particle sizes also affected the crystal size, the optimal for nucleation being 432 nm. Classical Nucleation Theory (CNT) can interpret surface chemistry effects but does not support the effect of particle size. This paper reports that the combined use of both geometrical and chemical interactions results in an increased ability to control the nucleation and growth of protein crystals.
The nucleation of protein crystals as a race against time with on- and off-pathways
Journal of Applied Crystallography, 2017
High supersaturation levels are a necessary but insufficient condition for the crystallization of purified proteins. Unlike most small molecules, proteins can take diverse aggregation pathways that make the outcome of crystallization assays quite unpredictable. Here, dynamic light scattering and optical microscopy were used to show that the nucleation of lysozyme crystals is preceded by an initial step of protein oligomerization and by the progressive formation of metastable clusters. Because these steps deplete the concentration of soluble monomers, the probability of obtaining protein crystals decreases as time progresses. Stochastic variations of the induction time are thus amplified to a point where fast crystallization can coexist with unyielding regimes in the same conditions. With an initial hydrodynamic radius of ∼100 nm, the metastable clusters also promote the formation of protein crystals through a mechanism of heterogeneous nucleation. Crystal growth (on-pathway) takes p...
Crystal Growth & Design, 2016
Major discoveries in structural biology depend on obtaining well-diffracting macromolecular crystals. This necessity has motivated many fundamental studies on protein crystallization using lysozyme as a model system. In the present contribution, we report the unprecedented observation of lysozyme crystals that stop dissolving under undersaturated conditions imposed to sub-microliter crystallization drops at mild temperatures. Subsequent growth of the same crystals is apparently undisturbed after the drops are cooled below the saturation temperature. The succession of heating/cooling cycles only partially recovers crystal dissolution while crystal growth becomes gradually slower. Ultimately, increasing and decreasing the temperature between 10 and 37 °C has no visible effect on the size of the crystals. We ascribe this phenomenon to the partial denaturation of the soluble protein in the drop as evidenced by the decreasing glycoside hydrolase activity of lysozyme over the incubation time. The disturbances in the phase transition processes are explained as the result of the changed chemical potential due to different folding states. In a time when the high-hanging fruits in structural biology have to be picked, the present findings call attention to interfacial phenomena as an important, though often imperceptible, aspect that affects protein stability and justifies further optimization of current crystallization methods.
Weakly-bound Dimers that Underlie the Crystal Nucleation Precursors in Lysozyme Solutions
2018
ABSTRACTProtein crystallization is central to understanding of molecular structure in biology, a vital part of processes in the pharmaceutical industry, and a crucial component of numerous disease pathologies. Crystallization starts with nucleation and how nucleation proceeds determines the crystallization rate and essential properties of the resulting crystal population. Recent results with several proteins indicate that crystals nucleate within preformed mesoscopic protein-rich clusters. The origin of the mesoscopic clusters is poorly understood. In the case of lysozyme, a common model of protein biophysics, earlier findings suggest that clusters exist owing to the dynamics of formation and decay of weakly-bound transient dimers. Here we present evidence of a weakly bound lysozyme dimer in solutions of this protein. We employ two electrospray mass spectrometry techniques, a combined ion mobility separation mass spectrometry and a high-resolution implementation. To enhance the weak...
A Model for Tetragonal Lysozyme Crystal Nucleation and Growth
Crystal Growth & Design, 2002
Macromolecular crystallization is a complex process, involving a system that typically has five or more components (macromolecule, water, buffer + counterion, and precipitant). Whereas small molecules have only a few contacts in the crystal lattice, macromolecules generally have 10's or even 100's of contacts between molecules. Formation of a consistent, ordered, three-dimensional (3D) structure may be difficult or impossible in the absence of any or presence of too many strong interactions. Further complicating the process is the inherent structural asymmetry of monomeric (single chain) macromolecules. The process of crystal nucleation and growth involves the ordered assembly of growth units into a defined 3D lattice. We propose that tetragonal lysozyme crystal nucleation and growth solutions are highly self-associated and that associated species having 4 3 helix symmetry are the building blocks for the nucleation process. This solution phase self-association carries over into the crystal growth phase, with the aggregated species as the growth units, recapitulating the nucleation process. The symmetry acquired in solution phase self-association facilitates both nucleation and crystal growth. If this model is correct, then fluids and crystal growth models assuming a strictly monodisperse nutrient solution need to be revised. This model has been developed from experimental evidence based upon face growth rate, atomic force microscopy, and fluorescence energy transfer data for the nucleation and growth of tetragonal lysozyme crystals.
Pre-assembled clusters distort crystal nucleation kinetics in supersaturated lysozyme solutions
Biophysical Chemistry, 2007
Efficient determination of three-dimensional protein structures is critical for unraveling structure-function relationships and for supporting targeted drug design. A major impediment to these efforts is our lack of control over the nucleation and growth of high-quality protein crystals for X-ray structure determinations. While basic research on protein crystal growth mechanisms has provided valuable new insights, studies of crystal nucleation have been plagued by inconsistent and outright contradictory results. Using dynamic light scattering and SDS gel electrophoresis, we have investigated possible causes of these inconsistencies. We find that commercial sources of lyophilized hen-egg white lysozyme (HEWL) used in nucleation studies contain significant populations of large (∼100 nm), pre-assembled lysozyme clusters that can readily evade standard assays of sample purity. In supersaturated solutions, these clusters act as heterogeneous nucleation centers that enhance the rate of crystal nucleation and significantly deteriorate the quality of macroscopic crystals.
Crystal Growth & Design, 2007
We observed two-dimensional (2D) nucleation behavior on {110} and {101} faces of tetragonal crystals of model protein lysozyme by laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM). We measured, for the first time directly and noninvasively, the 2D nucleation rates using 99.99% pure lysozyme, 98.5% pure lysozyme (Seikagaku Co.), and 99.99% pure lysozyme with intentionally added impure proteins (fluorescent-labeled lysozyme, covalently bonded dimer of lysozyme, and 18 kDa polypeptide). We found that 2D nucleation was the dominant growth mechanism under conditions adopted in this study, and the 2D nucleation occurred randomly on the entire crystal surface irrespective of supersaturation within the range of σ) ln(C/C e)) 0-1.4, where C is a bulk lysozyme concentration and C e the solubility (crystal size: 0.2-0.3 mm). Repeated 2D nucleation, which continued for 3-4 layers, was also observed mainly when the impure proteins were present. In addition, multilayered 2D islands were formed after the adsorption of relatively large foreign particles on the crystal surface. From the comparison between the 2D nucleation rates determined on the {110} faces with and without the impure proteins, we concluded that homogeneous 2D nucleation occurred under a higher supersaturation range (σ > 0.8), irrespective of the presence of the impurities. In contrast, under a lower supersaturation range (σ < 0.8), we found that significant heterogeneous 2D nucleation dominated the growth mainly when the impure proteins were present. The {101} faces exhibited more significant heterogeneous 2D nucleation induced by smaller amounts of impurities than in the case of the {110} faces. We also determined the ledge free energies of the homogeneous and heterogeneous nucleation. Within the experimental conditions used in this study, we could not find significant dependence of the ledge free energies of the heterogeneous nucleation on the kinds of impure proteins.
Do protein crystals nucleate within dense liquid clusters?
Acta Crystallographica Section F Structural Biology Communications, 2015
Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10−3of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear f...