A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.     

Trypsin crystallization by membrane-based techniques

To grow protein crystals is not an easy task; moreover, if we need to grow protein crystals with controlled shape, size, and size distribution, depending on their application, the mission becomes even harder. Membrane crystallization has been recognized as an interesting tool for growing protein crystals with enhanced crystallization kinetics, both in static and in forced solution flow configuration, without detrimental effects on crystal quality. In the present work, we have studied the membrane crystallization process of benzamidine inhibited trypsin from bovine pancreas (BPT), with ammonium sulphate (dissolved in Tris-HCl buffer, 0.1 M, pH 8.5), as precipitant agent. We have demonstrated that, by using the membrane crystallization technique, BPT crystals can be obtained in 24-48 h, in static configuration, and in 4-7 days, in a forced solution flow system, depending on the experimental conditions. Furthermore, the kinetics of BPT crystallization have been modulated, to control the morphological characteristics of the crystals produced, by an accurate selection of the operative parameters involved in the process. The active membrane surface and the flow rate of extraction solvent in quiescent configuration, and the solution velocity in forced convection solution experiments, were the parameters investigated. In this respect, membrane crystallization techniques have been assessed as an interesting way for growing proteins, and more specifically enzyme crystals, with high control on the final properties of the crystalline material produced, with potential fundamental implication in the field of structural biology and biotechnology.     

A simple apparatus for controlling nucleation and size in protein crystal growth

A simple device is described for controlling vapor equilibrium in macromolecular crystallization as applied to the protein crystal growth technique commonly referred to as the "hanging drop" method. Crystal growth experiments with hen egg white lysozyme have demonstrated control of the nucleation rate. Nucleation rate and final crystal size have been found to be highly dependent upon the rate at which critical supersaturation is approached. Slower approaches show a marked decrease in the nucleation rate and an increase in crystal size.     

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.     

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.     

Precipitants and additives for membrane crystallization of lysozyme

Membrane crystallization is a newly developed crystallization technique that has proven to be superior in producing good crystal forms under operating conditions that are not appropriate to perform the crystallization process by other traditional techniques. In this work, static membrane crystallization was carried out on lysozyme, with hollow-fiber microporous hydrophobic membranes. Numerous precipitant and additive types and concentrations were employed in the crystallization processes in order to select the most appropriate precipitant and additive types and to find their corresponding concentration levels that can yield the best crystal forms. The crystallization processes were analyzed in two ways: firstly, by evaluation of the transmembrane fluxes obtained by using different precipitants and additives; secondly, by utilization of the images and results obtained from the micrography and IR spectra in comparisons and evaluations of the crystals formed under all kinds of conditions. Moreover, the size distributions of the crystals yielded under several typical crystallization conditions were analyzed, and turbidity and induction time periods obtained during typical crystallization experiments were also measured. Amongst the numerous precipitants and additives tested, the most appropriate precipitant type and additive were chosen and their concentrations were optimized. Good lysozyme crystals were obtained using a certain precipitant and additive. The obtained results from this work further support the advantages of utilizing the membrane crystallization technique for macromolecule crystallizations.     

Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily

Here we report the successful three-dimensional crystallization of GlpT, the glycerol-3-phosphate transporter from Escherichia coli inner membrane. GlpT possesses 12 transmembrane alpha-helices and is a member of the major facilitator superfamily. It mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane. Approximately 20 phospholipid molecules per protein, identified as negatively charged phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, were required for the monodispersity of purified GlpT. Analytical size-exclusion chromatography proved to be efficient in identifying detergents for GlpT monodispersity. Nine such detergents were later used for GlpT crystallization. Screening for crystal nucleation was carried out with a variety of polyethylene glycols as the precipitant over a wide pH range. Subsequent identification of a rigid protein core by limited proteolysis and mass spectroscopy resulted in better-ordered crystals. These crystals exhibited order to 3.7 A resolution in two dimensions. However, the stacking in the third dimension was partially disordered. This stacking problem was overcome by using a detergent mixture and manipulating the ionic interactions in the crystallization solution. The resulting GlpT crystals diffracted isotropically to 3.3 A resolution and were suitable for structure determination by X-ray crystallography.     

In Situ μGISAXS: I. Experimental Setup for Submicron Study of Protein Nucleation and Growth

In this study, we used microbeam grazing-incidence small-angle x-ray scattering (μGISAXS) to investigate in situ protein nucleation and crystal growth assisted by a protein nanotemplate, and introduced certain innovations to improve the method. Our aim was to understand the protein nanotemplate method in detail, as this method has been shown to be capable of accelerating and increasing crystal size and quality, as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing protein crystals at different stages of nucleation and growth. Two model proteins, lysozyme and thaumatin, were used under unique flow conditions to differentially probe protein crystal nucleation and growth.     

An automated pipeline to screen membrane protein 2D crystallization

Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein–lipid–detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility.     

Pre-assembled clusters distort crystal nucleation kinetics in supersaturated lysozyme solutions

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 (approximately 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.     

Parameters for crystal growth of ribosomal subunits

A systematic analysis of the parameters that control the crystal growth of the large subunit of ribosomes from B stearothermophilus has been carried out. Several parameters have been identified and classified according to their significance. It was found that only biologically active particles can crystallize and that the critical period for the crystallization process is the first few days, during which changes in the volume and content of the crystallization drop are of importance for both nucleation and crystal growth. Consequently, an experimental procedure for fine control of these variables has been developed. As a result of these studies, the reproducibility of crystal formation was increased, and larger and more stable crystals have been obtained.     

A Technique for High-Throughput Protein Crystallization in Ionically Cross-Linked Polysaccharide Gel Beads for X-Ray Diffraction Experiments

A simple technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads has been developed for contactless handling of crystals in X-ray crystallography. The method is designed to reduce mechanical damage to crystals caused by physical contact between crystal and mount tool and by osmotic shock during various manipulations including cryoprotection, heavy-atom derivatization, ligand soaking, and diffraction experiments. For this study, protein crystallization in alginate and κ-carrageenan gel beads was performed using six test proteins, demonstrating that proteins could be successfully crystallized in gel beads. Two complete diffraction data sets from lysozyme and ID70067 protein crystals in gel beads were collected at 100 K without removing the crystals; the results showed that the crystals had low mosaicities. In addition, crystallization of glucose isomerase was carried out in alginate gel beads in the presence of synthetic zeolite molecular sieves (MS), a hetero-epitaxic nucleant; the results demonstrated that MS can reduce excess nucleation of this protein in beads. To demonstrate heavy-atom derivatization, lysozyme crystals were successfully derivatized with K₂PtBr₆ within alginate gel beads. These results suggest that gel beads prevent serious damage to protein crystals during such experiments.     

Room to move: crystallizing membrane proteins in swollen lipidic mesophases

The cubic phase or in meso crystallization method is responsible for almost 40 solved integral membrane protein structures. Most of these are small and compact proteins. A model for how crystals form by the in meso method has been proposed that invokes a transition between mesophases. In light of this model, we speculated that a more hydrated and open mesophase, of reduced interfacial curvature, would support facile crystallization of bigger and bulkier proteins. The proposal was explored here by performing crystallization in the presence of additives that swell the cubic phase. The additive concentration inducing swelling, as quantified by small-angle X-ray diffraction, coincided with a "crystallization window" in which two, very different transmembranal proteins produced crystals. That the swollen mesophase can grow structure-grade crystals was proven with one of these, the light-harvesting II complex. In most regards, the structural details of the corresponding complex resembled those of crystals grown by the conventional vapour diffusion method, with some important differences. In particular, packing density in the in meso-grown crystals was dramatically higher, more akin to that seen with water-soluble proteins, which accounts for their enhanced diffracting power. The layered and close in-plane packing observed has been rationalized in a model for nucleation and crystal growth by the in meso method that involves swollen mesophases. These results present a rational case for including mesophase-swelling additives in screens for in meso crystallogenesis. Their use will contribute to broadening the range of membrane proteins that yield to structure determination.     

Shape evolution and thermal stability of lysozyme crystals: effect of pH and temperature

The properties of crystalline protein materials are closely linked to crystal shape. However, the effective strategies for the shape control of protein crystals are lacking. The conventional sitting-drop vapor-diffusion method was employed to investigate the influence of pH and temperature on the crystal nucleation behavior of hen egg white lysozyme. Moreover, the size distributions of protein crystals grown at different conditions were analyzed. Differential scanning calorimetry was employed to evaluate the thermal stability of lysozyme crystals. The results indicated that pH and temperature will affect the supersaturation and electrostatic interactions among protein molecules in the nucleation process. In particular, the crystals with different aspect ratios can be selectively nucleated, depending upon the choice of pH and temperature. Therefore, this study provided a simple method for obtaining shape-controlled lysozyme crystals and supplied some information on thermal behaviors of lysozyme crystals grown at different pH values.     

Evaluation of a model for seeded isothermal batch protein crystallization

Bulk protein crystallization, unlike small molecule crystallization, has found very limited use in biopharmaceutical manufacture. Most work in this area targets obtaining single large crystals for molecular structure determination by crystallography. Design and optimization of bulk crystallization for protein recovery and purification is much less common, and requires a mathematical model for analysis of laboratory data suitable for scale-up purposes. Traditionally, the crystal size distribution and method of moments is used to characterize the crystallization process. A simpler method is presented in this paper that utilizes the desupersaturation curve. The method uses an approach that does not require expensive instrumentation or characterization of the seed crystal size distribution. The method is extended to allow determination of both the mass deposition rate constant and the growth rate order from a single desuperaturation curve. Experimental data for the bulk crystallization of ovalbumin are used to validate the method. The rate constants and rate order obtained using the new method compare well with literature values. Scale-up is illustrated by prediction of the impact of changes in seed mass on protein crystallization. This new method offers a straightforward and low-cost alternative to traditional methods for the analysis and scale-up of protein crystallization data.     

Crystallization and micro FT-IR spectroscopy investigation of cytochrome bc1 complex

A simple method to obtain large red crystals of cytochrome bc1 complex from beef heart mitochondria has been developed. These crystals are very stable. Their shapes are retained for a long time in tip-sealed Pasteur pipets placed in a refrigerator. The structure of crystalline cytochrome bc1 complex by micro FT-IR spectroscopy has been investigated. Based on the IR spectra of cytochrome c, the empirical assignments of the major infrared frequencies of cytochrome bc1 complex are given. Infrared frequencies and relative intensities of variable orientation and section of crystal are significantly different. These imply that infrared spectral characterization of the membrane protein crystallization is associated with the variable symmetries and orientations of the structure. Experimental results show that phospholipid exists in the crystal of cytochrome bc1 complex. The membrane protein is probably spanned on the mitochondrial membrane and buried in phospholipid bilayer in an asymmetric manner.     

The effect of temperature and solution pH on the nucleation of tetragonal lysozyme crystals.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.     

Electron crystallography of membrane proteins: two-dimensional crystallization and screening by electron microscopy

Structural and functional information of membrane proteins at ever-increasing resolution is being obtained by electron crystallography. While a large amount of work on the development of methods for electron microscopy and image processing has resulted in tremendous advances in terms of speed of data collection and resolution, general guidelines for crystallization are first starting to emerge. Yet two-dimensional crystallization itself will always remain the limiting factor of this powerful approach in structural biology. Two-dimensional crystallization through detergent removal by dialysis is the most widely used technique. Four main factors need to be considered for the dialysis method: the protein preparation, the detergent, the lipid added as well as any constituent lipid, and the buffer conditions. Equally important is proper and careful screening to identify two-dimensional crystals.     

Crystallization and micro FT-IR spectroscopy investigation of cytochrome bc_1 complex

A simple method to obtain large red crystals of cytochrome bc1 complex from beef heart mitochondria has been developed. These crystals are very stable. Their shapes are retained for a long time in tip-sealed Pasteur pipets placed in a refrigerator. The structure of crystalline cytochrome bc1 complex by micro FT-IR spectroscopy has been investigated. Based on the IR spectra of cytochrome c, the empirical assignments of the major infrared frequencies of cytochrome bc1 complex are given. Infrared frequencies and relative intensities of variable orientation and section of crystal are significantly different. These imply that infrared spectral characterization of the membrane protein crystallization is associated with the variable symmetries and orientations of the structure. Experimental results show that phospholipid exists in the crystal of cytochrome bc1 complex. The membrane protein is probably spanned on the mitochondrial membrane and buried in phospholipid bilayer in an asymmetric manner.     

A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.