Nucleation and growth of microbial lipase crystals from clarified concentrated fermentation broths

Bulk crystallization is emerging as a new industrial operation for protein recovery. Characterization of bulk protein crystallization is more complex than protein crystallization for structural study where single crystals are grown in flow cells. This is because both nucleation and crystal growth processes are taking place while the supersaturation falls. An algorithm is presented to characterize crystallization using the rates of the two kinetic processes, nucleation and growth. The values of these rates allow ready comparison of the crystallization process under different operating conditions. The crystallization, via adjustment to the isoelectric pH of a fungal lipase from clarified fermentation broth, is described for a batch stirred reactor. A maximum nucleation rate of five to six crystals formed per microliter of suspension per second and a high power dependency ( approximately 11) on the degree of supersaturation were found. The suspended protein crystals were found to grow at a rate of up to 15-20 nm/s and also to exhibit a high power dependency ( approximately 6) of growth rate on the degree of supersaturation.     

Practical physics behind growing crystals of biological macromolecules

The aim of this review is to provide biocrystallographers who intend to tackle protein-crystallization with theory and practical examples. Crystallization involves two separate processes, nucleation and growth, which are rarely completely unconnected. Here we give theoretical background and concrete examples illustrating protein crystallization. We describe the nucleation of a new phase, solid or liquid, and the growth and transformation of existing crystals obtained by primary or secondary nucleation or by seeding. Above all, we believe that a thorough knowledge of the phase diagram is vital to the selection of starting position and path for any crystallization experiment.     

Lattice simulations of protein crystal formation

A new algorithm is presented for the lattice simulation of protein crystal growth. The algorithm allows the calculation of the size distribution of microcrystals in the volume and timescale of experiments and within the framework of the previously-published microscopic model [A.M. Kierzek, W.M. Wolf, P. Zielenkiewicz, Biophys. J. 73 (1997) 571-580]. Simulations for the tetragonal lysozyme crystal show that there are two critical sizes in the development of ordered phase. The first one corresponds to the size of the smallest stable complex which, in the case of the tetragonal lysozyme crystal, is the particular tetramer. In a volume of 5 mul the tetramer appears in the millisecond timescale. The second critical radius of approximately 100 monomers is only reached by a few of all the smallest stable complexes formed in the solution. The model predicts that out of 10(7) tetramers which appear in solution, only eight reach the size of 100 monomers within 8 h. After exceeding the second critical radius the microcrystals grow to the size of 10(4) monomers in the minute timescale and are thus assumed to quickly lead to macroscopic crystals. The predicted number of crystals formed during 8 h of nucleation is in qualitative agreement with arrested nucleation experiments.     

Analysis and statistics of crystallisation success increase by composition modification of protein and precipitant mixing ratio

The nucleation zone has to be reached for any crystal to grow, and the search for crystallization conditions of new proteins is a trial and error process. Here a convenient screening strategy is studied in detail that varies the volume ratio of protein sample to the reservoir solution in the drop to initiate crystallization that is named "composition modification". It is applied after the first screen and has been studied with twelve proteins. Statistical analysis shows a significant improvement in screening using this strategy. The average improvement of "hits" at different temperatures is between 32 and 42%, for examples, 41.8% ± 14.0% and 35.7% ± 12.4% (± standard deviation) at 288 K and 300 K, respectively. Remarkably, some new crystals were found by composition modification which increased the probability of reaching the nucleation zone to initiate crystallization. This was confirmed by a phase diagram study. It is also demonstrated that composition modification can further increase crystallisation success significantly (1.3 times) after the improvement of "hits" by temperature screening. The trajectories of different composition modifications during vapour diffusion were plotted, further demonstrating that protein crystallizability can be increased by hitting more parts of the nucleation zone. It was also found to facilitate the finding of initial crystals for proteins of low solubility. These proteins gradually become more concentrated during the vapour diffusion process starting from a larger protein solution ratio in the initial mixture.     

Crystallization of IgG1 by mapping its liquid-liquid phase separation curves

Monoclonal antibody therapeutics is an important and fast expanding market. While production of these molecules has been a major area of research, much less is known regarding the stabilization of these proteins for delivery as drugs. Crystallization of antibodies is one such promising route for protein stabilization at high titers, and here we took a systematic approach to initiate crystallization through nucleation in a simple PEG (polyethylene glycol), protein in water solution. A ternary mixture of globular proteins, PEG, and water will undergo a liquid-liquid phase separation (LLPS) as shown in a phase diagram or a Binodal curve. Of particular interest within the phase diagram is the position of the critical point, which is where nucleation occurs most rapidly. Detailed LLPS maps were created by increasing concentrations of PEG (from 5% to 11%) and IgG (from 1 to 20 mg/mL). By increasing the molecular weight (MW) of PEG (and hence its radius of gyration) from 1,000 to 6,000 g/mol, the temperatures of the critical point of nucleation were shown to increase. Once these curves were determined, nucleation experiments were conducted close to a chosen critical point (10.5 mg/mL IgG in 11% PEG 1000) and after 3 weeks, crystals of IgG of approximately 100 microm in size were successfully formed. This is the first example of crystallization of an antibody through systematic mapping of LLPS curves, which is a fundamental step towards the scale-up of antibody crystallization.     

Protein nucleation and crystallization by homologous protein thin film template

A new method of protein nucleation and crystallization based on Langmuir-Blodgett technology is here utilized for the template stimulation of crystal growth of so far non-crystallized proteins. Microcrystals (60-120 microm) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method. The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in an earlier study with a model system (chicken egg white lysozyme) as an unexpected acceleration and enhancement in the crystal growth.     

Ionic liquids as selectors for controlling the crystallization nucleation of hen egg white lysozyme

Ionic liquids (ILs) have biomaterial applications and are used for protein crystallization. The effect of two imidazolium-based ILs, 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl) and 1,3-dimethylimidazolium iodide ([dmim]I), on the nucleation kinetics of lysozyme was investigated by determining the nucleation induction time, and nucleation parameters were evaluated. The values of interfacial tension calculated for solutions with added 30 g/L ILs [C₄mim]Cl and [dmim]I, and without added ILs were 99.03, 109.7, and 107.3 mJ/m², respectively. Compared with solutions without IL addition, the critical free energy change, size, and molecular number of critical nuclei decreased and the nucleation rate increased after the addition of [C₄mim]Cl. In contrast, the critical free energy change, size, and molecular number of critical nuclei increased and the nucleation rate decreased after the addition of [dmim]I. These new findings provide insights into controlling lysozyme crystallization separation, and present ILs as potentially useful additives for controlling the crystallization of macromolecules.     

Predicting protein crystallizability and nucleation

The outcome of protein crystallization attempts is often uncertain due to inherent features of the protein or to the crystallization process that are not fully under control of the experimentalist. The aim of this contribution is to propose user-friendly tools that can increase the success rate of a protein crytallization project. Different bioinformatic approaches to predict the crystallization feasibility (before any crystallization attempts are undertaken) are discussed and a novel approach to assess the nucleation process of a given protein is proposed. Practical examples illustrate these two points.     

Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation

Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, "in cubo", has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field.     

Using nanoliter plugs in microfluidics to facilitate and understand protein crystallization

Protein crystallization is important for determining protein structures by X-ray diffraction. Nanoliter-sized plugs--aqueous droplets surrounded by a fluorinated carrier fluid--have been applied to the screening of protein crystallization conditions. Preformed arrays of plugs in capillary cartridges enable sparse matrix screening. Crystals grown in plugs inside a microcapillary may be analyzed by in situ X-ray diffraction. Screening using plugs, which are easily formed in PDMS microfluidic channels, is simple and economical, and minimizes consumption of the protein. This approach also has the potential to improve our understanding of the fundamentals of protein crystallization, such as the effect of mixing on the nucleation of crystals.     

Monomer-dependent secondary nucleation in amyloid formation

Secondary nucleation of monomers on the surface of an already existing aggregate that is formed from the same kind of monomers may lead to autocatalytic amplification of a self-assembly process. Such monomer-dependent secondary nucleation occurs during the crystallization of small molecules or proteins and self-assembled materials, as well as in protein self-assembly into fibrous structures. Indications of secondary nucleation may come from analyses of kinetic experiments starting from pure monomers or monomers supplemented with a low concentration of pre-formed aggregates (seeds). More firm evidence requires additional experiments, for example those employing isotope labels to distinguish new aggregates arising from the monomer from those resulting from fragmentation of the seed. In cases of amyloid formation, secondary nucleation leads to the formation of toxic oligomers, and inhibitors of secondary nucleation may serve as starting points for therapeutic developments. Secondary nucleation displays a high degree of structural specificity and may be enhanced by mutations or screening of electrostatic repulsion.     

The 1.45 A three-dimensional structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus

The conversion of solar radiation to chemical energy by photosynthetic organisms provides the primary driving force for life on earth. Light energy is captured by a variety of pigments, usually bound to proteins, which vary with different types of organisms. We report here the 1.45 A resolution three-dimensional structure of one such pigment protein, C-phycocyanin, from Synechococcus elongatus. The structure is at the highest resolution achieved for any such phycobiliprotein. This level of resolution was made possible by implementing a novel crystallization method whereby nucleation is decoupled from subsequent growth, by incubating crystallizing drops for 7h under nucleation conditions and then transferring them to metastable conditions for growth. This is done without touching the crystallization drops throughout the process.     

Ice nucleation inhibition: mechanism of antifreeze by antifreeze protein

The effect of antifreeze protein type III (one type of fish antifreeze protein) on ice crystallization was examined quantitatively based on a "micro-sized ice nucleation" technique. It was found for the first time that antifreeze proteins can inhibit the ice nucleation process by adsorbing onto both the surfaces of ice nuclei and dust particles. This leads to an increase of the ice nucleation barrier and the desolvation kink kinetics barrier, respectively. Based on the latest nucleation model, the increases in the ice nucleation barrier and the kink kinetics barrier were measured. This enables us to quantitatively examine the antifreeze mechanism of antifreeze proteins for the first time.     

杂质对蛋白质晶体影响研究进展

综述了杂质对蛋白质晶体生长影响研究领域的进展情况. 对可能的杂质来源以及杂质对结晶过程的影响进行了介绍.重点介绍了和结晶蛋白质分子结构相似的杂质分子的影响, 包括晶体成核、生长形态、表面形貌、生长动力学、质量等,以及杂质在晶体中的重新分配.     

Surface Exposed Free Cysteine Suppresses Crystallization of Human γD-Crystallin

Human γD-crystallin (HGD) has remarkable stability against condensation in the human lens, sometimes over a whole lifetime. The native protein has a surface exposed free cysteine that forms dimers (Benedek, 1997; Ramkumar et al., 1864)^1,2 without specific biological function and leads to further protein association and/or aggregation, which creates a paradox for understanding its stability. Previous work has demonstrated that chemical modification of the protein at the free cysteine (C110), increases the temperature at which liquid–liquid phase separation occurs (LLPS), lowers protein solubility and suggests an important role for this amino acid in maintaining its long-term resistance to condensation. Here we demonstrate that mutation of the cysteine does not alter the structure or solubility (liquidus) line for the protein, but dramatically increases the protein crystal nucleation rate following LLPS, suggesting that the free cysteine has a vital role in suppressing crystallization in the human lens.     

Heterogeneous nucleation of protein crystals on fluorinated layered silicate

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface.     

Nucleation of Polymorphic Amyloid Fibrils

One and the same protein can self-assemble into amyloid fibrils with different morphologies. The phenomenon of fibril polymorphism is relevant biologically because different fibril polymorphs can have different toxicity, but there is no tool for predicting which polymorph forms and under what conditions. Here, we consider the nucleation of polymorphic amyloid fibrils occurring by direct polymerization of monomeric proteins into fibrils. We treat this process within the framework of our newly developed nonstandard nucleation theory, which allows prediction of the concentration dependence of the nucleation rate for different fibril polymorphs. The results highlight that the concentration dependence of the nucleation rate is closely linked with the protein solubility and a threshold monomer concentration below which fibril formation becomes biologically irrelevant. The relation between the nucleation rate, the fibril solubility, the threshold concentration, and the binding energies of the fibril building blocks within fibrils might prove a valuable tool for designing new experiments to control the formation of particular fibril polymorphs.     

SAmBA: An interactive software for optimizing the design of biological macromolecules crystallization experiments

SAmBA is a new software for the design of minimal experimental protocols using the notion of orthogonal arrays of strength 2. The main application of SAmBA is the search of protein crystallization conditions. Given a user input defining the relevant effectors/variables (e.g., pH, temperature, salts) and states (e.g., pH: 5, 6, 7 and 8), this software proposes an optimal set of experiments in which all tested variables and the pairwise interactions between them are symmetrically sampled. No a priori restrictions on the number and range of experimental variables is imposed. SAmBA consists of two complementary programs, SAm and BA, using a simulated annealing approach and a backtracking algorithm, respectively. The software is freely available as C code or as an interactive JAVA applet at http://igs-server.cnrs-mrs.fr . Proteins 29:252–257, 1997. © 1997 Wiley-Liss, Inc.     

Crystallization mechanisms of hemoglobin C in the R state

Crystallization of the mutated hemoglobin, HbC, which occurs inside red blood cells of patients expressing betaC-globin and exhibiting the homozygous CC and the heterozygous SC (in which two mutant beta-globins, S and C, are expressed) diseases, is a convenient model for processes underlying numerous condensation diseases. As a first step, we investigated the molecular-level mechanisms of crystallization of this protein from high-concentration phosphate buffer in its stable carbomonoxy form using high-resolution atomic force microscopy. We found that in conditions of equilibrium with the solution, the crystals' surface reconstructs into four-molecule-wide strands along the crystallographic a (or b) axis. However, the crystals do not grow by the alignment of such preformed strands. We found that the crystals grow by the attachment of single molecules to suitable sites on the surface. These sites are located along the edges of new layers generated by two-dimensional nucleation or by screw dislocations. During growth, the steps propagate with random velocities, with the mean being an increasing function of the crystallization driving force. These results show that the crystallization mechanisms of HbC are similar to those found for other proteins. Therefore, strategies developed to control protein crystallization in vitro may be applicable to pathology-related crystallization systems.     

蛋白质结晶的研究进展

概括了蛋白质结晶的基本过程,阐述了蛋白质结晶的早期发展历程,重点介绍了蛋白质结晶的近期研究状况,主要包括:形核机理的研究、结晶条件的筛选和结晶技术的优化以及基于结构的药物设计技术。特别是对离子液体在蛋白质结晶过程中的应用及发展前景进行了讨论。