Protein Crystal Growth Research Articles

Wet-Stamped Precipitant Gradients Control the Growth of Protein Microcrystals in an Array of Nanoliter Wells

Wet stamping initiated from a hydrogel reservoir soaked in a salt precipitant sets-up multiple conditions for the growth of protein crystals over an array of nanoliter wells. Diffusive delivery of the precipitant translates into time- and space-varying fluxes of the precipitant in the wells. These fluxes, in turn, control the numbers of seeded crystals as well as their dimensions. Reaction-diffusion equations with a nucleation term are developed to model the process of crystal growth and to relate the “schedules” of precipitant delivery to the dimensions and habits of the protein microcrystals in the array.

Shape-Control of Protein Crystals in Patterned Microwells

This Communication demonstrates how selectively functionalized, patterned microscale wells can be used as small-volume (fL) crystallization vessels to grow protein crystals. This surface-patterned approach enables proteins to be crystallized with well-controlled shapes and uniform sizes from 3 μm down to 600 nm. Three different proteinslysozyme, thaumatin, and glucose isomerasewere crystallized as model systems to demonstrate the generality of this technique. Different polymorphs were formed simply by controlling the evaporation conditions.

High-quality Protein Crystal Growth under Space Environment

High-quality Protein Crystal Growth under Space Environment

New developments in crystal growth from solutions: Oxides, proteins, and nitrides

Abstract Controlling the nucleation and growth processes is important for growing large, high-quality crystals of oxides, proteins, and nitrides from a solution. We must understand how the solution conditions should be controlled, and determine a universal principle that is applicable to the growth of various high-quality crystals. In this report, we will present the effects of solution stirring and nucleation control on the solution growth of CsLiB 6 O 10 (CLBO), proteins, and GaN crystals with respect to the following issues: (1) effect of solution stirring on the growth of high-quality CLBO crystals, (2) high-quality protein crystal growth by means of forced nucleation induced by laser irradiation and the solution-stirring method, and (3) high-quality 2-in GaN crystal by Na flux liquid-phase epitaxy method.

Can Small Laboratories Do Structural Genomics?

Structural genomics centers have often been regarded as the “members only” clubs associated with expensive equipment, material, and personnel. Although much expense has been invested worldwide in structural genomics consortia, there has been an enormous amount of criticism on the apparent disproportional output produced relative to the amount of money spent. Here, we review and highlight some of the major achievements in structural genomics projects coupled to X-ray crystallography. Structural genomics has advanced technologies in high throughput bioinformatics, cloning and recombinant protein expression, crystal growth, and crystallographic structure determination. If cost can be reduced and feasibility increased then a modest-sized laboratory research group can significantly contribute to structural genomics efforts. We demonstrate this by examining a sequenced and annotated hyperthermophilic archaeal genome (about 2 million base pairs) with our own structural genomics methods. By incorporating proven strategies developed from large structural genomic centers with practical innovations, we have constructed a mini-pipeline in which a small group consisting of as few as two people can survey 1500 open reading frames for cloning, expression, crystallization, and structure determination for less than $200 000.

Crystallogenesis Research for Biology in the Last Two Decades as Seen from the International Conferences on the Crystallization of Biological Macromolecules

The series of 11 International Conferences on the Crystallization of Biological Macromolecules (ICCBM) took place over the period 1986–2006 in the USA (four times), Germany (two times), China, France, Japan, Spain, and lastly the 11th in Canada in Quebec City. Here we review the first 10 ICCBMs. Their focus was to bring rational approaches to the field of protein crystal growth and thus overcome the rate-limiting step in macromolecular X-ray crystallography. This survey summarizes how the ICCBM series contributed to the emergence of the science of biocrystallogenesis. This was achieved through the joint efforts of scientists from the small molecule crystal growth community and from biochemists, biophysicists, and protein crystallographers. Highlights from each conference are discussed, and scientific synergies are emphasized. While the first conferences focused on fundamentals, especially from the standpoint of physics and biochemical considerations, the more recent conferences stressed applications in structural biology, to advanced methods of crystallization, and of crystal quality improvement. Particular attention will be given to themes that were recurrent through all the ICCBMs: purity and impurities, solution properties of macromolecules under precrystallization conditions, microgravity and assessment of crystal quality, as well as specific trends of practical interest to structural biology.

Using Microfluidics to Decouple Nucleation and Growth of Protein Crystals

A high throughput, low volume microfluidic device has been designed to decouple the physical processes of protein crystal nucleation and growth. This device, called the Phase Chip, is constructed out of poly(dimethylsiloxane) (PDMS) elastomer. One of the Phase Chip's innovations is to exploit surface tension forces to guide each drop to a storage chamber. We demonstrate that nanoliter water-in-oil drops of protein solutions can be rapidly stored in individual wells thereby allowing the screening of 1000 conditions while consuming a total of only 10 mug protein on a 20 cm(2) chip. Another significant advance over current microfluidic devices is that each well is in contact with a reservoir via a dialysis membrane through which only water and other low molecular weight organic solvents can pass, but not salt, polymer, or protein. This enables the concentration of all solutes in a solution to be reversibly, rapidly, and precisely varied in contrast to current methods, such as the free interface diffusion or sitting drop methods, which are irreversible. The Phase Chip operates by first optimizing conditions for nucleation by using dialysis to supersaturate the protein solution, which leads to nucleation of many small crystals. Next, conditions are optimized for crystal growth by using dialysis to reduce the protein and precipitant concentrations, which leads small crystals to dissolve while simultaneously causing only the largest ones to grow, ultimately resulting in the transformation of many small, unusable crystals into a few large ones.

Vibration-induced particle drift in a fluid cell under microgravity

A theoretical investigation into the effect of small vibrations on the behaviour of a small particle contained in a fluid cell under microgravity is presented. Diffusion-controlled material processing such as protein crystal growth can be adversely affected by small vibrations called g-jitter, if a relative motion is induced between the particle and surrounding fluid. When a fluid cell containing a small particle such as a protein crystal is vibrated parallel to the wall nearest to the particle, the particle oscillates with a certain amplitude and a hydrodynamic force in the direction normal to the wall is induced. Theoretical models based on an inviscid fluid assumption are used to predict the particle amplitude variation and drifting motion. Due to an external vibration such as g-jitter, the oscillating particle is predicted to drift towards the wall and the particle oscillation amplitude to decrease slightly as the distance between the particle and wall is reduced. The reduction in particle ampitude also depends on the particle-to-fluid density ratio. The particle drift towards the nearest wall acclerates due to an increasing attraction force, and the drifting speed increases with both the vibration frequency and particle diameter. Even for small protein crystals with a density close to that of the fluid, the time required to drift from the center of the fluid cell to the wall is predicted to be much shorter than the growth time.

PEG-Induced Morphologically Unstable Growth of Tetragonal Hen Egg-White Lysozyme Crystals

Poly(ethylene glycol) (PEG) is an often used crystallizing agent in screening experiments for protein crystallization conditions. To investigate the influence of poly(ethylene glycol)-4600 on the diffusion process during protein crystal growth, a series of in situ optical microscopy experiments were performed in which the diffusion coefficient was changed by the addition of PEG. Depending on the balance between diffusion coefficient and driving force, lysozyme crystals were found to grow faceted, kinetically rough, or morphologically unstable. In situ observations show the development of a lysozyme crystal from polyhedral stability to instability and back to stability again as the balance changes within one experiment. A stability diagram showing the occurrence of morphological instability based on the starting conditions is presented, showing that kinetic roughening stabilizes the crystal morphology. The results are interpreted in terms of a competition between two-dimensional nucleation at the edges of the crystal and step flow on its surface. In this, surface protein concentration profiles derived from observed crystal shapes are used.

On the protein crystal formation as an interface-controlled process with prototype ion-channeling effect.

A superdiffusive random-walk action in the depletion zone around a growing protein crystal is considered. It stands for a dynamic boundary condition of the growth process and competes steadily with a quasistatic, curvature-involving (thermodynamic) free boundary condition, both of them contributing to interpret the (mainly late-stage) growth process in terms of a prototype ion-channeling effect. An overall diffusion function contains quantitative signatures of both boundary conditions mentioned and indicates whether the new phase grows as an orderly phase or a converse scenario occurs. This situation can be treated in a quite versatile way both numerically and analytically, within a generalized Smoluchowski framework. This study can help in (1) elucidating some dynamic puzzles of a complex crystal formation vs biomolecular aggregation, also those concerning ion-channel formation, and (2) seeing how ion-channel-type dynamics of non-Markovian nature may set properly the pace of model (dis)ordered protein aggregation.

A down-to-Earth approach

In seeking out ideal conditions for growing protein crystals, solutions have increasingly been found in the low-gravity conditions of space. But answers might be lurking in fields closer to home.

Design, Synthesis, and Performance of NTA-modified Lipids as Templates for Histidine-tagged Protein Crystallization

Abstract Current methods of drug discovery and development typically involve either structure-based drug design approaches or high-throughput screening methods for lead identification. The structure-based discovery process relies heavily upon the labor-intensive determination of three-dimensional protein structures using X-ray diffraction (XRD) and NMR techniques. Although tens of thousands of soluble protein structures have been resolved at the atomic level using these methods, there are many drug targets, particularly integral membrane proteins, whose structures have not been elucidated. In the case of XRD, this limitation often arises from the lack of reliable and predictable methods for protein crystallization. The extensive number of proteins crystallized by the interfacial templating approach suggests that this method may offer great promise for structural biology applications, particularly for proteins that do not readily form X-ray quality crystals from bulk solution. This review highlights the various approaches that have been used for templating of protein crystallization at gas–liquid and solid–liquid interfaces. Special emphasis is placed on the synthesis of NTA- and IDA-conjugated lipids, their use in templating and concentrating proteins at lipid monolayer interfaces, and protein structure elucidations that have been facilitated by these metal-chelating lipids. Current approaches to control the nucleation and growth of protein crystals involving microfluidics, dip pen nanolithography, and non-covalent symmetry-based templating approaches are also discussed. Taken together, the progress in this field suggests that the interfacial templating approach to crystallization can develop into a powerful tool for high-throughput structural analysis by providing a universal, readily controllable method for crystallizing proteins.

From egg to crystal

A practical hands-on course encompassing enzyme purification, biochemical characterization, and crystallization that completed the course work of 350 second-year bachelor students enrolled in molecular biology/biochemistry was given at the Université Louis Pasteur of Strasbourg (France). The experimental part of the practical dealt entirely with the model protein lysozyme isolated from hen egg-white. It was designed as a research project to give students the possibility to practice biochemical methods such as chromatography, electrophoresis, and spectrophotometry. It also included enzyme activity assay and protein crystal growth that are usually taught in master-level courses. The organization of the practical work and the related experimental procedures are described and discussed.

On the Slow Kinetics of Protein Crystallization

Although they require surprisingly high supersaturations, both nucleation and growth of protein crystals proceed substantially more slowly as compared to small molecule crystallization. The slow nucleation of the protein crystals is explained by the steric restriction for association of the protein molecules that is due to their highly inhomogeneous surfaces. Over their surfaces, the proteins exhibit a limited number of discrete patches that are the only attractive portions on the molecule. A simple model for protein crystal nucleation has been devised on this basis, which enables calculations of nucleus form, energy barrier, and probability for nucleus formation. It turns out that the unusually high supersaturations used experimentally are necessary for the formation of relatively small nuclei, because the probability for bigger fluctuations is negligibly small. On the other hand, the big size of the protein molecules contributes additionally for the slow growth of the protein crystals. It causes huge Burgers vectors of the dislocations. In turn, this leads to a locally increased chemical potential and an effective decrease of the local supersaturation at the step source.

Magnetically controlled gravity for protein crystal growth

The occurrence of convective flows during crystal growth is believed to adversely affect crystal quality. Space-based crystal growth is therefore actively pursued, particularly for protein crystals, because buoyancy-driven convection is suppressed in microgravity. Here the authors demonstrate that magnetic fields can be used to tune the effective gravity from 1to−0.15g during the growth of diamagnetic lysozyme crystals and that convection can be damped, stopped, and even reversed. The growth velocity is strongly reduced in simulated microgravity. This method provides a versatile and accessible way to realize an earth-based tunable gravity environment for crystal growth in which protein crystal quality may be optimized.

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 (∼ 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.

Development of a Superconducting Magnet for High Magnetic Force Field Application

Laboratory size superconducting magnets to generate a magnetic force field of 1500 T <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> /m in a 40 mm and 50 mm diameter room temperature bore have been developed. In order to generate such a high magnetic force field, a superconducting magnet is combined with main coils and a reverse coil. The magnet can be operated in persistent mode and the field decay is less than 1 ppm/h, which means that the sample can be kept in a micro gravity environment for more than two years. This magnet will be used in various applications in the field of micro gravity application, such as protein crystal growth.

Probabilistic approach to protein crystal nucleation

AbstractSimple probabilistic model for step‐wise growth of polymers is used for making some parallels with the nucleation of protein crystals. Although the considerations are made within 1D case, this approximation still shows some important peculiarities of protein crystal nucleation and growth. Thus, the present approach turns out to be useful for interpretation of some important experimental results regarding these processes. (© 2007 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Nonequivalence of second virial coefficients from sedimentation equilibrium and static light scattering studies of protein solutions

Experimental data for ovalbumin and lysozyme are presented to highlight the nonequivalence of second virial coefficients obtained for proteins by sedimentation equilibrium and light scattering. Theoretical considerations confirm that the quantity deduced from sedimentation equilibrium distributions is B 22, the osmotic second virial coefficient describing thermodynamic nonideality arising solely from protein self-interaction. On the other hand, the virial coefficient determined by light scattering is shown to reflect the combined contributions of protein–protein and protein–buffer interactions to thermodynamic nonideality of the protein solution. Misidentification of the light scattering parameter as B 22 accounts for published reports of negative osmotic second virial coefficients as indicators of conditions conducive to protein crystal growth. Finally, textbook assertions about the equivalence of second virial coefficients obtained by sedimentation equilibrium and light scattering reflect the restriction of consideration to single-solute systems. Although sedimentation equilibrium distributions for buffered protein solutions are, indeed, amenable to interpretation in such terms, the same situation does not apply to light scattering measurements because buffer constituents cannot be regarded as part of the solvent: instead they must be treated as non-scattering cosolutes.

Protein crystal growth in gels and stationary magnetic fields

AbstractThaumatin, lysozyme, and ferritin single crystals were grown in solutions and gels without and with surrounding strong stationary magnetic fields. The crystal size, number and alignment in dependence on the induction force were analysed. The crystal quality, like mosaicity, as function of the magnetic force is discussed by using synchrotron X‐ray diffraction analysis. (© 2007 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)