Protein Crystallization Conditions Research Articles

Surface-mutagenesis strategies to enable structural biology crystallization platforms.

A key prerequisite for the successful application of protein crystallography in drug discovery is to establish a robust crystallization system for a new drug-target protein fast enough to deliver crystal structures when the first inhibitors have been identified in the hit-finding campaign or, at the latest, in the subsequent hit-to-lead process. The first crucial step towards generating well folded proteins with a high likelihood of crystallizing is the identification of suitable truncation variants of the target protein. In some cases an optimal length variant alone is not sufficient to support crystallization and additional surface mutations need to be introduced to obtain suitable crystals. In this contribution, four case studies are presented in which rationally designed surface modifications were key to establishing crystallization conditions for the target proteins (the protein kinases Aurora-C, IRAK4 and BUB1, and the KRAS-SOS1 complex). The design process which led to well diffracting crystals is described and the crystal packing is analysed to understand retrospectively how the specific surface mutations promoted successful crystallization. The presented design approaches are routinely used in our team to support the establishment of robust crystallization systems which enable structure-guided inhibitor optimization for hit-to-lead and lead-optimization projects in pharmaceutical research.

A 3D-Printed Standardized Modular Microfluidic System for Droplet Generation

Droplet-based microfluidics has a variety of applications, such as material synthesis and single-cell analysis. In this paper, we propose a modular microfluidic system using projection micro-stereolithography three-dimensional (3D) printing technology for droplet generation. All modules are designed using a standard cubic structure with a specific leakage-free connection interface. Versatile droplets, including single droplets, alternating droplets, merged droplets, and Janus particles, have been successfully produced. The droplet size and the generation rate can be flexibly controlled by adjusting the flow rates. The influence of the flow rate fraction between the discrete phase and the continuous phase over the generation of the alternating and merged droplets is discussed. Furthermore, the 'UV curing' module can be employed to solidify the generated droplets to avoid coalescence and fix the status of the Janus particles. The proposed modular droplet generators are promising candidates for various chemical and biological applications, such as single-cell incubation, screening of protein crystallization conditions, synthesis of nanoparticles, and gene delivery. In addition, we envision that more functional modules, e.g., valve, microreactor, and detection modules, could be developed, and the 3D standardized modular microfluidics could be further applied to other complex systems, i.e., concentration gradient generators and clinical diagnostic systems.

Structural Exploration on Palmitoyltransferase DHHC3 from Homo sapiens.

DHHC3 belongs to a family of DHHC palmitoyltransferase, which catalyzes the S-palmitoylation of target proteins by attaching a fatty acyl group to a cysteine. Recently, DHHC3 has been demonstrated to be a promising antitumor target in cancer therapeutics. However, the detailed structure and catalysis mechanism of DHHC3 remain elusive, considering its sequence diversity from the DHHC homologues with known crystal structures. Here, we described the expression and purification of human DHHC3 (hDHHC3) and truncated hDHHC3 with the flexible N-terminal domain (NTD) removed. Purified hDHHC3 proteins were used under various conditions for protein crystallization. LAMTOR1, one of the interacting proteins of hDHHC3 to facilitate the crystallization, was further identified by mass spectrometry and co-immunoprecipitation assay. The structural exploration using cryogenic electronic microscopy (cryo-EM) on the inactive hDHHS3 mutant showed a typical sideview of membrane proteins. These results provide a preliminary guidance for the structural determination of DHHC3.

Protein Crystallization in a Microfluidic Contactor with Nafion®117 Membranes.

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

Easy-to-Use Osmosis-Based Microfluidic Chip for Protein Crystallization: Application to a Monoclonal Antibody

We developed a disposable microfluidic chip which mimics the vapor diffusion method for exploring protein crystallization conditions but at the nanoliter scale. This device exploits the permeation of water through a thin poly(dimethylsiloxane) (PDMS) layer separating droplets stored in the chip and containing a mixture of proteins and precipitants from an open microfluidic reservoir. The water chemical activity fixed by the reservoir makes it possible to modify the volume of the droplets in a controlled way (reduction or increase). Because PDMS is only permeable to water, the imposed water activity therefore increases or decreases the concentration of the solutes present in the droplet and consequently the supersaturation of the solution. The specificity of our approach, in addition to the low volumes ensuring controlled mass transport conditions, is that the concentration of all the solutes is known at any time, thus allowing the extraction of quantitative information on the crystallization process. We exploit these chips on a protein of therapeutic interest: the full-length monoclonal antibody anti-CD20. Our experiments allow us in particular to estimate both the solubility of this protein and the width of the metastable zone with Na2SO4 and PEG400 as crystallizing agents. We also show that the fine-tuning of the permeation rate makes it possible to perform crystallization/dissolution cycles to selectively dissolve small crystals and increase the mean size of the remaining anti-CD20 crystals.

Nanoliter-scale liquid metering and droplet generation based on a capillary array for high throughput screening

Herein, we developed a simple approach for quantitative metering of nanoliter-scale liquids in parallel based on a capillary array and applied it in high throughput screening protein crystallization conditions. The quantitative metering of liquids was achieved by using capillary force to spontaneously introduce the liquids into short capillaries with fixed length and inner diameter, and the nanoliter-scale droplets were generated by using a pneumatic pump to deliver liquids out from the capillary channels. We adopted measures of sharpening the capillary tips and performing a hydrophobic treatment on the tip surface to significantly reduce the capillary residues during the liquid aspirating and dispensing process, and thus improved the precision to 0.2%–3.5% relative standard deviations (RSD, n = 3) in metering droplets in the range of 280 pL-90 nL. We evaluated the performance of the system in metering liquids of different surface tensions and viscosity. On the basis of this approach, we built a capillary array system with 12 capillaries, by which parallel generation of 12 nL droplets of 12 samples could be achieved in 40 s with a relative standard deviation (RSD) of 1.2%. We applied the system in the screening of lysozyme crystallization conditions of 48 precipitants with 7.5 nL precipitant and 7.5 nL protein solutions in each crystallization droplet reactor, to demonstrate its potentials in large-scale high-throughput screening and analysis with different samples.

Octamers participation in the formation of lysozyme ordered layers from crystallization solutions

The results of the study of thin film ordered protein structures formed from polydisperse solutions of lysozyme using Langmuir technology are presented. The proposed method for producing protein films is based on a modification of the Langmuir-Schaeffer method, which consists in using a pre-prepared protein solution with the addition of a precipitant. The pre-prepared protein solutions’ parameters (protein and precipitant concentrations, buffer type, etc.) correspond to protein crystallization conditions. It is assumed that protein oligomers formed in the solution as a result of the addition of the precipitant (in particular, for lysozyme these oligomers include octamers) are directly involved in the formation of Langmuir protein layers on the surface of the liquid and on solid substrates. Using the method of grazing-incidence X-ray standing waves, the structure of multilayered protein systems formed from polydisperse solutions was studied, which made it possible to determine directly the position of precipitant ions (NaCl, CuCl2 and NiCl2) relative to the protein layer. The method of processing the X-ray reflectivity and grazing-incidence X-ray standing waves data, based on the use of information on the atomic structure of lysozyme octamers isolated from the crystal lattice, made it possible to determine the thickness and electron density of protein films and to reveal the orientation of protein molecules in the layer.

High-Throughput Differential Scanning Fluorimetry of GFP-Tagged Proteins.

Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure-activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal perturbation effects on GFP fluorescence. As a result of this unique principle, DSF-GTP can specifically measure the thermal stability of a target protein in the presence of other proteins. Additionally, the GFP provides a unique in-assay quality control measure. Here, we describe the workflow, steps, and important considerations for executing a DSF-GTP experiment in a 96-well plate format.

Diamond B23 Beamline for Synchrotron Radiation Circular Dichroism (SRCD): High Throughput CD (HTCD) and CD Imaging (CDi) Applications

High throughput (HTCD) using 96 and 384 multiplate for the optimisation of protein crystallization conditions, protein-ligand binding screening, and CD Imaging (CDi) of thin films of materials (proteins, nucleic acids, carbohydrates, polymers with embedded drugs, organic LED, and in general any chiral material with or without achiral substrates) are the results of the unique highly collimated microbeam light generated at B23 beamline at Diamond Light Source in the vacuum UV to visible wavelength range (130-650nm). B23 is the only bespoke beamline worldwide to characterise the structural and conformational properties of biologically important molecules using micro-devices unattainable with bespoke bench-top CD instruments. Recent applications and latest beamline upgrades will be discussed.

Improved protein-crystal identification by using 2,2,2-trichloroethanol as a fluorescence enhancer

The identification of initial lead conditions for successful protein crystallization is crucial for structural studies using X-ray crystallography. In order to reduce the number of false-negative conditions, an emerging number of fluorescence-based methods have been developed which allow more efficient identification of protein crystals and help to distinguish them from salt crystals. Detection of the native tryptophan fluorescence of protein crystals is one of the most widely used methods. However, this method can fail owing to the properties of the crystallized protein or the chemical composition of the crystallization trials. Here, a simple, fast and cost-efficient method employing 2,2,2-trichloroethanol (TCE) has been developed. It can be performed with a standard UV-light microscope and can be applied to cases in which detection of native tryptophan fluorescence fails. In four test cases this method had no effect on the diffraction properties of the crystals and no structural changes were observed. Further evidence is provided that TCE can be added to crystallization trials during their preparation, making this method compatible with high-throughput approaches.

Applicability of Probabilistic Nucleation Modelling for the Analysis of Microfluidics Data

Microfluidics tools have been developing rapidly over the past decade, as they offer unparalleled ability for controlling nucleation and tracking crystallisation events inside very large numbers of individual nanolitre-size droplets. They have demonstrated a significant potential for screening protein crystallization conditions and for the direct determination of inorganic products solubility curves. The accepted basis for analysing microfluidics data is the probabilistic nucleation model originally proposed by Pound and La Mer (1952). Given the significance of this model for the purpose of analysing microfluidics data, the paper conducts a review of its hypotheses, usage and applicability. A step-by-step derivation of the model equations confirms that the time variation of the proportion of empty droplets which microfluidics experiments can provide with high accuracy is indeed the recommended method for estimation of nucleation kinetic parameters from microfluidics experiments. The paper shows that, depending on its implementation, the model predicts different rates of appearance of crystals inside individual droplets. The paper focuses on two distinct implementation modes, referred to as constant supersaturation and single nucleation event modes. By confronting model prediction with microfluidics measurements for eflucimibe in octanol, the paper finds that both modes yield different model predictions, shedding light on the potential and limits of the probabilistic nucleation model for the analysis of microfluidics data.

Size and Shape Controlled Crystallization of Hemoglobin for Advanced Crystallography

While high-throughput screening for protein crystallization conditions have rapidly evolved in the last few decades, it is also becoming increasingly necessary for the control of crystal size and shape as increasing diversity of protein crystallographic experiments. For example, X-ray crystallography (XRC) combined with photoexcitation and/or spectrophotometry requires optically thin but well diffracting crystals. By contrast, large-volume crystals are needed for weak signal experiments, such as neutron crystallography (NC) or recently developed X-ray fluorescent holography (XFH). In this article, we present, using hemoglobin as an example protein, some techniques for obtaining the crystals of controlled size, shape, and adequate quality. Furthermore, we describe a few case studies of applications of the optimized hemoglobin crystals for implementing the above mentioned crystallographic experiments, providing some hints and tips for the further progress of advanced protein crystallography.

From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RCvir). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RCvir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.

Enhanced Crystallization of Lysozyme Mediated by the Aggregation of Inorganic Seed Particles

We show that aggregation plays a major role in seeded growth of protein crystals. The seeded batch approach provides the opportunity to set the starting conditions for protein crystallization by adding a defined amount of well-characterized seed particles. The experimental observations for tetragonal hen egg-white lysozyme (LSZ) confirm the concept of the oriented aggregation of larger building blocks to form a protein crystal. It was shown that the aggregation of the seed particles/bioconjugates is advantageous for the product quality in terms of larger and more defined LSZ crystals and in terms of accelerated reaction kinetics. We present a population balance (PB) model for the seeded batch crystallization of LSZ considering the aggregation of growth units to form protein crystals. For the modeling of crystal growth, evolving particle size distributions (PSDs) of agglomerating LSZ molecules were measured by dynamic light scattering (DLS). Moreover, the aggregation of seed particles in LSZ solutions unde...

Recent progress in protein and protein-related systems using DSC techniques

As one family of the most important biomoleclues, proteins have gained more and more attention since the day they were discovered and the trend is still growing. Even so, many problems are emerging in this filed and thus new techiniques and ideas are required for solving these problems. In this regard, differential scanning calorimetry (DSC) is a conventional but useful method to obtain the relationship between heat capacity and temperature of peoteins. In the past decade, new ideas have been applied to DSC and new methods have been developed. This is reflected in this minireview. First, important progresses have been made on protein unfloding or folding mechanisms. A new method called “interruption-incubation protocol” was developd to judge wheather a protein unfolds in a two-state mechanism or not. Compared to the conventional van’t Hoff analysis method, the new approach breaks the limitations of old method on the requirement of protein properties, namely the reversibility of protein folding and the equilibrium assumption during the unfoding process. Also, this method avoids the inaccuracies of enthalpy change for protein unfolding caused by improper baseline determination. Thus, the “interruption-incubation protocol” has a lower possibility of misjudgement and turns out to be an effective way for analysing “two-state or non-two state” proteins. In addition, by combining DSC with phase transition models instead of chemical equilibrium assumption, several theories were proposed which can provide a rough landscape of protein folding or unfolding process. Among these theories, the “variable barrier model” is an interesting one, which can directly give the barrier heights in protein folding as long as the barrier heights are smaller than ~4 RT . Second, a new method was proposed to derive protein stability curve directly by combining DSC with isothermal chemical denaturation (ICD) method. This method targets those “problematic” proteins, when the denaturation process is not perfect reversible. Three cases were discussed, namely the unreliability of protein Δ C p determination, the irreversibility of protein thermal denaturation and the extreme stability of specific proteins. For the first case, a more reliable protein temperature-dependent Δ C p was obtained by a combantion of DSC and ICD method, which was used for further calculation of Δ G of the unfolding process. For the second case, the unfolding process of some proteins under low pH is reversible and protein stability result from DSC was highly consistent with that obtained by ICD method. Then, protein stability under other conditions such as high pH, where the denaturation would be irreversible, could be estimaed by ICD method. For the third case, a combination of several denaturants were used to obtain the protein stability curve. In addition, DSC can also be applied to optimize conditions for protein crystallization. Third, the application scope of DSC is expanded to complex protein-realted systems such as amyloid fibers or even biological samples like blood plasma. For amyloid fibers, DSC can provide useful information of their thermodynamic properties which can lead us to a better understanding of amyloid fiber formation; for biological samples, DSC thermograms can be used as “fingerprints” for clinical diagnosis since the features of DSC thermograms for healthy and sick individuals are different. To summarize, DSC is a powerful tool to obtain protein properties and will play a more important role in the future.

Sans Studies of Bacteriorhodopsin Incorporation and Crystallization in Cubic Phase

The direct crystallization of membrane proteins from lipidic mesophases has become an increasingly important, and even routinely used, tool in the field of membrane protein structural biology. Despite the method's practical successes, however, the exact molecular mechanisms for crystallization remain poorly understood. It has been proposed that protein molecules first align in transient, locally lamellar regions of lipid, forming small 2D crystals that initiate growth. These crystals could then stack to form the 3D crystal, which is fed laterally by lamellar-phase membrane extensions from the bulk cubic phase. Nevertheless, the initial formation of 2D crystal “patches” and initiation of lamellar transition has not been experimentally observed. Other mechanisms of crystal formation can also be envisioned, and the possibility of multiple crystallization mechanisms exists. A clear understanding of these mechanisms would allow for the rational optimization of crystallization conditions for different proteins, including the use of different lipids and additives.Using Small-Angle Neutron Scattering (SANS), we have begun a systematic study of cubic phase crystallization using the membrane protein Bacteriorhodopsin (bR) in monoolein cubic phase as a model system, alone and in the presence of precipitants. SANS with contrast variation is uniquely suited for the study of proteins in cubic phase, since the scattering contribution of the large excess of lipid can be eliminated, allowing the scattering from embedded protein to be directly measured without interference. This allows us to study bR embedded in the monoolein cubic phase.

CrystPro: Spatiotemporal Analysis of Protein Crystallization Images.

Thousands of experiments corresponding to different combinations of conditions are set up to determine the relevant conditions for successful protein crystallization. In recent years, high throughput robotic set-ups have been developed to automate the protein crystallization experiments, and imaging techniques are used to monitor the crystallization progress. Images are collected multiple times during the course of an experiment. Huge number of collected images make manual review of images tedious and discouraging. In this paper, utilizing trace fluorescence labeling, we describe an automated system called CrystPro for monitoring the protein crystal growth in crystallization trial images by analyzing the time sequence images. Given the sets of image sequences, the objective is to develop an efficient and reliable system to detect crystal growth changes such as new crystal formation and increase of crystal size. CrystPro consists of three major steps- identification of crystallization trials proper for spatio-temporal analysis, spatio-temporal analysis of identified trials, and crystal growth analysis. We evaluated the performance of our system on 3 crystallization image datasets (PCP-ILopt-11, PCP-ILopt-12, and PCP-ILopt-13) and compared our results with expert scores. Our results indicate a) 98.3% accuracy and .896 sensitivity on identification of trials for spatio-temporal analysis, b) 77.4% accuracy and .986 sensitivity of identifying crystal pairs with new crystal formation, and c) 85.8% accuracy and 0.667 sensitivity on crystal size increase detection. The results show that our method is reliable and efficient for tracking growth of crystals and determining useful image sequences for further review by the crystallographers.

Developments in the Implementation of Acoustic Droplet Ejection for Protein Crystallography.

Acoustic droplet ejection (ADE) enables crystallization experiments at the low-nanoliter scale, resulting in rapid vapor diffusion equilibration dynamics and efficient reagent usage in the empirical discovery of structure-enabling protein crystallization conditions. We extend our validation of this technology applied to the diverse physicochemical property space of aqueous crystallization reagents where dynamic fluid analysis coupled to ADE aids in accurate and precise dispensations. Addition of crystallization seed stocks, chemical additives, or small-molecule ligands effectively modulates crystallization, and we here provide examples in optimization of crystal morphology and diffraction quality by the acoustic delivery of ultra-small volumes of these cofactors. Additional applications are discussed, including set up of in situ proteolysis and alternate geometries of crystallization that leverage the small scale afforded by acoustic delivery. Finally, we describe parameters of a system of automation in which the acoustic liquid handler is integrated with a robotic arm, plate centrifuge, peeler, sealer, and stacks, which allows unattended high-throughput crystallization experimentation.

A centrifugal microfluidic device for screening protein crystallization conditions by vapor diffusion

This paper describes a centrifuge-based microfluidic device for performing nanoliter-scale protein crystallization trials by vapor diffusion. By combining the capillary effect and centrifugal force, this device spontaneously and parallelly meters and dispenses protein samples and precipitants into 24 reaction chambers for crystallization screening. Furthermore, the design of two-level capillary stop valves and vapor-diffusion chambers in this device allows vapor transport between the protein droplet and the stored precipitant, perfectly equivalent to that observed in conventional vapor diffusion techniques, which slowly brings the protein droplet into a supersaturation state to nucleate and grow protein crystals. We demonstrate the power of this device by testing it for rapid screening of protein crystallization conditions. Compared with the conventional hanging-drop technique, this device can identify more crystallization conditions and provide a wider range of supersaturation rates, while using two orders of magnitude less protein sample. This centrifuge-based microfluidic device provides a simple and low-cost tool for individual laboratories to rapidly screen initial protein crystallization conditions.

Generation of two-dimensional concentration-gradient droplet arrays on a two-layer chip for screening of protein crystallization conditions

We developed a two-layer microfluidic chip for the generation of two-dimensional concentration-gradient droplet arrays. All of the channels, including a concentration-gradient formation channel network, an oil phase channel network, and droplet generators, were ingeniously arranged on a single polydimethylsiloxane (PDMS) substrate, which facilitates the fabrication of the chip. Ninety-six lines of subnanoliter droplets with concentration gradients can be formed on a 6-inlet PDMS chip. With the aqueous phase flow rate of 1.0 μL/min and the oil phase flow rate of 35 μL/min, relative standard deviation of the droplet sizes in the same channel was below 5.0 %, while in the 96 channels was 7.0 %. Multiparameter screening of protein crystallization conditions was demonstrated by using the two-dimensional concentration-gradient droplet arrays. Protein, precipitant, and buffer each at 16 concentrations for lysozyme crystallization were tested simultaneously in one 6-inlet chip, and five precipitants each at eight concentrations were screened at the same time in one 10-inlet chip. The concentration-gradient droplet chip proved to be a useful tool for screening crystallization conditions, characterized by low consumption, high throughput, convenience, and automatization.