Formation Of Protein Crystals Research Articles

An Analysis of Protein Crystals Grown under Microgravity Conditions

Microgravity has been shown to be an excellent tool for protein crystal formation. A retrospective analysis of all publicly available crystallization data, including many that have not yet been published, clearly demonstrates the value of the microgravity environment for producing superior protein crystals. The parameters in the database (the Butler Microgravity Protein Crystal Database, BμCDB) that were evaluated pertain to both crystal morphology and diffraction quality. Success metrics were determined as improvements in size, definition, uniformity, mosaicity, diffraction quality, resolution limits, and B factor. The proteins in the databases were evaluated by molecular weight, protein type, the number of subunits, space group, and Mattew’s Coefficient. Compared to ground experiments, crystals grown in a microgravity environment continue to show improvement across all metrics evaluated. General trends as well as numerical differences are included in the assessment of the BμCDB. The microgravity environment improves crystal formation across a spectrum of metrics and the datasets utilized for this investigation are excellent tools for this evaluation.

Convolutional neural network approach for the automated identification of in cellulo crystals.

In cellulo crystallization is a rare event in nature. Recent advances that have made use of heterologous overexpression can promote the intracellular formation of protein crystals, but new tools are required to detect and characterize these targets in the complex cell environment. The present work makes use of Mask R-CNN, a convolutional neural network (CNN)-based instance segmentation method, for the identification of either single or multi-shaped crystals growing in living insect cells, using conventional bright field images. The algorithm can be rapidly adapted to recognize different targets, with the aim of extracting relevant information to support a semi-automated screening pipeline, in order to aid the development of the intracellular protein crystallization approach.

Controlling Protein Crystallization by Free Energy Guided Design of Interactions at Crystal Contacts

Protein crystallization can function as an effective method for protein purification or formulation. Such an application requires a comprehensive understanding of the intermolecular protein–protein interactions that drive and stabilize protein crystal formation to ensure a reproducible process. Using alcohol dehydrogenase from Lactobacillus brevis (LbADH) as a model system, we probed in our combined experimental and computational study the effect of residue substitutions at the protein crystal contacts on the crystallizability and the contact stability. Increased or decreased contact stability was calculated using molecular dynamics (MD) free energy simulations and showed excellent qualitative correlation with experimentally determined increased or decreased crystallizability. The MD simulations allowed us to trace back the changes to their physical origins at the atomic level. Engineered charge–charge interactions as well as engineered hydrophobic effects could be characterized and were found to improve crystallizability. For example, the simulations revealed a redesigning of a water mediated electrostatic interaction (“wet contact”) into a water depleted hydrophobic effect (“dry contact”) and the optimization of a weak hydrogen bonding contact towards a strong one. These findings explained the experimentally found improved crystallizability. Our study emphasizes that it is difficult to derive simple rules for engineering crystallizability but that free energy simulations could be a very useful tool for understanding the contribution of crystal contacts for stability and furthermore could help guide protein engineering strategies to enhance crystallization for technical purposes.

The fabrication of protein microbubbles with diverse gas core and the novel exploration on the role of interface introduction in protein crystallization

Recently, microbubbles with diameters ranging from a few to tens of microns have an increasing attractive attention in various applications. For microbubbles, exploring convenient and reliable fabrication approach is crucial, in particular achieving stable microbubbles filled with diverse gas. In this work, the baffled high intensity agitation (BHIA) cell was utilized to generate bovine serum albumin (BSA)-coated microbubbles. The characteristics of generated air-filled microbubbles was explored by adjustment of operating parameters. It was found that the microbubble size mainly depends on employed agitation speed. Microbubbles filled with different gas, such as N2, O2, and CO2, also were successfully obtained and show good stability at room temperature due to the formation of thick and elastic BSA shell. It was interestingly observed that a large number of regular BSA particles scattered around the trajectory of microbubble interfaces by scanning electron microscopy, which was obviously different from the particles formed from BSA solution in absence of microbubbles. The mechanism behind the phenomenon was investigated, and it was found that the abundant gas-liquid interface introduced by microbubbles is responsible for triggering the oriented aggregation of BSA molecules. The founding may reveal a spur significant for the effect of gas-liquid interface on the formation of protein crystals.

Kinetic Model of the Formation of Protein Crystals in Capillaries by Counterdiffusion

A kinetic model of crystallization of lysozyme is proposed, and computer calculations of this process are carried out. The conditions for the formation of such crystals are determined. Under these conditions, individual crystals were grown that were suitable for X-ray examination. The developed model enables prediction of the quantity, size, and place of crystal nucleation inside the capillary.

Cryo-EM reveals how protein crystals grow

Crystallizing a protein can be a shot in the dark. No one quite knows how protein crystals form, so researchers frequently must use trial and error to find the right crystallization conditions. Now, scientists have used cryo-electron microscopy (cryo-EM) to watch the earliest stages of protein crystal formation (Nature 2018, DOI: 10.1038/nature25971). What they learned allowed them to generate one desired crystal form, or polymorph, selectively, which could be handy for protein structure determination or in protein drug formulation. Crystallization starts with tiny clusters of protein molecules that act as seeds, says Mike Sleutel of Vrije Universiteit Brussels, who led the work. The clusters are too small to be seen with a light microscope, and they’re unstable. To address both challenges, Sleutel, Alexander E. S. Van Driessche of Grenoble Alps University, and colleagues rapidly froze their protein samples to halt molecular motion and got snapshots at various time points during

Optimizing Protein Crystal Formation of a Heterocyclization Domain from Yersiniabactin Synthetase

The bacterium Yersinia pestis, known for causing the bubonic plague, is an organism which must scavenge free iron from its host to grow. One method of obtaining iron is by producing a siderophore, yersiniabactin (Ybt), which is a secondary metabolite synthesized by a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS). This system utilizes three interesting heterocyclization (Cy) domains to introduce three five‐membered rings that are important for iron affinity. This research project aims to determine the molecular features of the third Cy domain (Cy3) that determine its function using X‐ray crystallography.Support or Funding InformationFunding for this research was provided in part by the Oracle Education Foundation grant to the College of Science and Mathematics and also by an ASBMB Undergraduate Research Award. Special thanks to the MIT crystallography facility. This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM123425‐01 to D.P.D.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Langmuir-Blodgett nanotemplates for protein crystallography.

The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the air-water interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 1-10 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods.

Berkeley Screen: a set of 96 solutions for general macromolecular crystallization.

Using statistical analysis of the Biological Macromolecular Crystallization Database, combined with previous knowledge about crystallization reagents, a crystallization screen called the Berkeley Screen has been created. Correlating crystallization conditions and high-resolution protein structures, it is possible to better understand the influence that a particular solution has on protein crystal formation. Ions and small molecules such as buffers and precipitants used in crystallization experiments were identified in electron density maps, highlighting the role of these chemicals in protein crystal packing. The Berkeley Screen has been extensively used to crystallize target proteins from the Joint BioEnergy Institute and the Collaborative Crystallography program at the Berkeley Center for Structural Biology, contributing to several Protein Data Bank entries and related publications. The Berkeley Screen provides the crystallographic community with an efficient set of solutions for general macromolecular crystallization trials, offering a valuable alternative to the existing commercially available screens.

The nucleation of protein crystals as a race against time with on- and off-pathways

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 place in parallel with cluster growth (off-pathway). The Janus-faced influence of the mesoscopic clusters is beneficial when it accelerates the formation of the first precrystalline nuclei and is detrimental as it depletes the solution of protein ready to crystallize. Choosing the right balance between the two effects is critical for determining the success of protein crystallization trials. The results presented here suggest that a mild oligomerization degree promotes the formation of a small number of metastable clusters which then catalyze the nucleation of well differentiated crystals.

The nanoscience behind the art of in-meso crystallization of membrane proteins.

The structural changes occurring at the nanoscale level within the lipid bilayer and driving the in-meso formation of large well-diffracting membrane protein crystals have been uniquely characterized for a model membrane protein, intimin. Importantly, the order to order transitions taking place within the bilayer and the lipidic nanostructures required for crystal growth have been shown to be general, occurring for both the cubic and the sponge mesophase crystallization pathways. For the first time, a transient fluid lamellar phase has been observed and unambiguously assigned for both crystallization pathways, present at the earliest stages of protein crystallogenesis but no longer observed once the crystals surpass the size of the average lyotropic liquid crystalline domain. The reported time-resolved structural investigation provides a significantly improved and general understanding of the nanostructural changes taking place within the mesophase during in-meso crystallization which is a fundamental advance in the enabling area of membrane protein structural biology.

Elucidating the fundamental forces in protein crystal formation: the case of crambin.

Molecular simulations of proteins have been usually accomplished through empirical or semi-empirical potentials, due to the large size and inherent complexity of these biological systems. On the other hand, a theoretical description of proteins based on quantum-mechanical methods would however provide an unbiased characterization of their electronic properties, possibly offering a link between these and the ultimate biological activity. Yet, such approaches have been historically hindered by the large amount of requested computational power. Here we demonstrate the feasibility of periodic all-electron density functional theory calculations in the description of the crystal of the protein crambin (46 aminoacids), which is determined with exceptional structural accuracy. We have employed the hybrid B3LYP functional, coupled to an empirical description of London interactions (D*) to simulate the crambin crystal with an increasing amount of lattice water molecules in the cell (up to 172H2O per cell). The agreement with the experiment is good for both protein geometry and protein-water interactions. The energetics was computed to predict crystal formation energies, protein-water and protein-protein interaction energies. We studied the role of dispersion interactions which are crucial for holding the crambin crystal in place. B3LYP-D* electrostatic potential and dipole moment of crambin as well as the electronic charge flow from crambin to the solvating water molecules (0.0015e per H2O) have also been predicted. These results proved that quantum-mechanical simulations of small proteins, both free and in their crystalline state, are now feasible in a reasonable amount of time, by programs capable of exploiting high performance computing architectures, allowing the study of protein properties not easily amenable through classical force fields.

Sigmoid kinetics of protein crystal nucleation

A non-linear differential equation expressing the new phase nucleation rate in the different steps of the process (non-stationary and stationary nucleation and in the plateau region) is derived from basic principles of the nucleation theory. It is shown that one and the same sigmoid (logistic) function describes both nucleation scenarios: the one according to the classical theory, and the other according to the modern two-stage mechanism of protein crystal formation. Comparison to experimental data on both insulin crystal nucleation kinetics and on bovine β-lactoglobulin crystallization indicates a good agreement with the sigmoidal prediction. Experimental data for electrochemical nucleation and glass crystallization obey the same sigmoid time dependence, and suggest universality of this nucleation kinetics law.

The amino-terminal structure of human fragile X mental retardation protein obtained using precipitant-immobilized imprinted polymers.

Flexibility is an intrinsic property of proteins and essential for their biological functions. However, because of structural flexibility, obtaining high-quality crystals of proteins with heterogeneous conformations remain challenging. Here, we show a novel approach to immobilize traditional precipitants onto molecularly imprinted polymers (MIPs) to facilitate protein crystallization, especially for flexible proteins. By applying this method, high-quality crystals of the flexible N-terminus of human fragile X mental retardation protein are obtained, whose absence causes the most common inherited mental retardation. A novel KH domain and an intermolecular disulfide bond are discovered, and several types of dimers are found in solution, thus providing insights into the function of this protein. Furthermore, the precipitant-immobilized MIPs (piMIPs) successfully facilitate flexible protein crystal formation for five model proteins with increased diffraction resolution. This highlights the potential of piMIPs for the crystallization of flexible proteins.

Serial femtosecond X-ray diffraction of in vivo crystals in intact yeast cells

Peroxisomes are membrane-enclosed organelles in eukaryotic cells with important roles in lipid metabolism and the scavenging of reactive oxygen species. Peroxisomes are capable of carrying an unusually high load of proteins, which under appropriate nutrient conditions results in the in situ crystallization of peroxisomal proteins in several yeast species and vertebrate hepatocytes [1,2]. In the methylotrophic yeast H.polymorpha, the predominant peroxisomal protein alcohol/methanol oxidase (AO) oligomerizes into octameric assemblies with a molecular mass of 600 kDa that spontaneously form 200-500 nm crystallites within peroxisomes [1]. We exposed H.polymorpha cell suspensions containing peroxisome-confined AO crystallites to femtosecond X-ray pulses at the Coherent X-ray Imaging (CXI) experimental endstation at the Linac Coherent Light Source. Peak detection routines mining the resulting scattering profiles identified >5000 Bragg-sampled diffraction patterns, providing the proof of concept that background scattering from the cells does not deteriorate the signal-to-noise ratio to an extent precluding observation of diffraction from individual AO crystallites. Summation patterns assembled from the individual frames match low-resolution powder diffraction patterns from concentrated suspensions of purified peroxisomes collected at the P14 beamline at the PETRAIII synchrotron, confirming that the observed diffraction mainly results from Bragg scattering of peroxisomal crystallites. To the best of our knowledge our data are the first to report room temperature X-ray diffraction from functional protein crystals in their native cellular environment. Currently the maximum resolution achieved in the diffraction patterns is limited to 20-15 Å. Future work will need to address improved sample preparation protocols in order to assess whether diffraction to a resolution sufficient to permit structure solution can be obtained. Protein crystal formation in vivo has been observed under physiological or pathological conditions in a number of other systems [3]. We hope that our results will help to establish serial femtosecond X-ray diffraction (SFX) as a method for structural characterization of cellular structures with crystalline content and provide a proof of concept for using in situ crystallization of proteins as a means to generate nanocrystalline samples for SFX.

高磁気力環境を利用した 高効率・高品位タンパク質結晶生成システムの開発とその利用

This review provides an outline of the “high-efficiency/high-quality protein crystal formation system” project. Precise determination of protein structures provides significant information for R&D on drugs and enzymes. Protein crystals of high integrity grown with high efficiency can accelerate such R&D. At present, a condition suitable for a certain protein crystal growth can only be found after several thousands of different conditions are examined by changing various parameters such as solvents, coexisting materials, temperature, pH and so on. It is considered that the convection of solution prevents the formation of good-quality crystal. Therefore, the control of gravity force effect through large magnetic force may be one of the potential parameters for protein crystal growth because it should contribute to suppressing the convection. To exert large magnetic force on protein solution or crystals equivalent with gravity force, high magnetic fields using a superconducting magnet are required. Observation of the crystallization process is important for efficient crystal formation, but it is difficult to carry out in-situ optical observation inside the superconducting magnet bore due to the existence of the high magnetic field. To facilitate efficient protein crystal growth under magnetic force, we developed a microscopic optical observation apparatus for multiple samples placed in a high magnetic field. The system consists of a superconducting magnet, crystallization plates and an optical periscope. The superconducting magnet used for this system is a high magnetic force generation type. The developed crystallization plates can contain 24 different protein solutions and can be stacked on each other. The 3D controllable periscope is made mainly from feeble magnetic materials that can be operated in high magnetic fields.

Characterization of salt interferences in second-harmonic generation detection of protein crystals

Studies were undertaken to assess the merits and limitations of second-harmonic generation (SHG) for the selective detection of protein and polypeptide crystal formation, focusing on the potential for false positives from SHG-active salts present in crystallization media. The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples. Out of 19 salts investigated, six produced significant background SHG and 15 of the 96 wells of a sparse-matrix screen produced SHG upon solvent evaporation. SHG-active salts include phosphates, hydrated sulfates, formates and tartrates, while chlorides, acetates and anhydrous sulfates resulted in no detectable SHG activity. The identified SHG-active salts produced a range of signal intensities spanning nearly three orders of magnitude. However, even the weakest SHG-active salt produced signals that were several orders of magnitude greater than those produced by typical protein crystals. In general, SHG-active salts were identifiable through characteristically strong SHG and negligible two-photon-excited ultraviolet fluorescence (TPE-UVF). Exceptions included trials containing either potassium dihydrogen phosphate or ammonium formate, which produced particularly strong SHG, but with residual weak TPE-UVF signals that could potentially complicate discrimination in crystallization experiments using these precipitants.

Development of a Protein Crystal Formation System With a Superconducting Magnet

We have developed a protein crystal formation system, which can, on the ground, suppress convections, exerting magnetic forces on the solution including proteins. The protein formation system consists of a superconducting magnet system, protein crystallization cells, and observational equipment; the latter two devices are placed in the magnet bore. The superconducting magnet system includes two coil groups, each generating a magnetic field opposing in direction to each other. The product of magnetic flux density and its spatial gradient is proportional to the magnetic force and needs to be approximately 1400 T2/m in order to levitate water. The magnet is operated in persistent current mode. The crystallization cells are made of a transparent plastic on a plate. The experimental space and cells can be controlled between 4°C and 20°C. The observational equipment, which is a kind of periscope, enables in-situ observation of protein solution in high magnetic fields. In order to optimize experimental conditions, we have also carried out a simulation study based on a solution flow model.

A flow simulation study of protein solution under magnetic forces

We have developed a superconducting magnet system generating magnetic forces able to compensate gravity and suppress convection of diamagnetic protein solution from which protein crystals precipitate. A simulation model has been proposed to elucidate the motion of protein solutions and search for the optimal conditions of the crystal formation process. This model incorporates general, non-uniform magnetic forces as external forces, while the previous models involve only simple, uniform magnetic forces. The simulation results indicate that the vertical component can suppress the convection of protein solution, while the horizontal component induces minimal convection. We, therefore, need to take into account the both components when considering the formation of protein crystals under magnetic forces.

Use of Dual Polarization Interferometry as a Diagnostic Tool for Protein Crystallization

The use of dual polarization interferometry (DPI) as a tool for probing the different possible outcomes of protein crystallization experiments is described. DPI is a surface analytical technique used for the characterization of structure and interactions of molecular layers on an optical waveguide surface for a wide range of applications, including protein-protein interactions and conformational changes. The application of this technique provides a "signature" of crystallization events, thus predicting if there will be protein crystal formation, amorphous precipitate, or clear solution. The technique was demonstrated on a number of model proteins, and it also produced meaningful results in the case of two problematic target proteins. DPI in conjunction with a dialysis setup, allows changes in the protein solution above the waveguide surface to be monitored simultaneously with continuous control of its precipitant content. DPI has the potential to be used as a powerful method for discovering crystallization conditions, for obtaining information on the crystallization process, and as an aid in crystal optimization. It has also provided what is, to the best of our knowledge, the most direct observation to date of salting-in behavior in a protein-salt solution.