Promote Protein Crystallization Research Articles

Methods for Engineering Binders to Multi-Pass Membrane Proteins.

Numerous potential drug targets, including G-protein-coupled receptors and ion channel proteins, reside on the cell surface as multi-pass membrane proteins. Unfortunately, despite advances in engineering technologies, engineering biologics against multi-pass membrane proteins remains a formidable task. In this review, we focus on the different methods used to prepare/present multi-pass transmembrane proteins for engineering target-specific biologics such as antibodies, nanobodies and synthetic scaffold proteins. The engineered biologics exhibit high specificity and affinity, and have broad applications as therapeutics, probes for cell staining and chaperones for promoting protein crystallization. We primarily cover publications on this topic from the past 10 years, with a focus on the different formats of multi-pass transmembrane proteins. Finally, the remaining challenges facing this field and new technologies developed to overcome a number of obstacles are discussed.

Fabrication of Hierarchical Bioinspired Superstructures Bearing Different Charges and Tunable Ability to Promote Protein Crystallization

Fabrication of Hierarchical Bioinspired Superstructures Bearing Different Charges and Tunable Ability to Promote Protein Crystallization

Protein crystallization with DNA templates

Protein crystallization plays a significant role in three-dimensional structural analysis and protein purification. It is important to increase the crystallization efficiency, which is possible by adding heterogeneous templates in crystallization systems. DNA is biologically compatible and artificially designable polymer, which is easy to extract. In this study, single- and double-stranded DNA of precise sequences were designed and used as templates to promote protein crystallization of lysozyme and catalase. Influence of DNA, single-stranded DNA with 10, 20, 40 bases and double-stranded DNA with 10, 20, 40 base pairs, were investigated. The success rate of obtaining crystals of lysozyme and catalase in equal period was significantly improved with the addition of DNA comparing without templates added. Double-stranded DNA led to higher nucleation rate than that with single-stranded DNA. The promotion of nucleation was more obvious at low concentration of protein solution and with longer chain DNA templates. Crystal number and crystallization rate was enhanced with addition of long double-stranded DNA templates. All the results confirm that DNA is an effective polymer additive to enhance protein crystallization, especially for the application of the scarce protein crystallization.

Interactions Between Peptide Assemblies and Proteins for Medicine

AbstractPeptide assembly is attractive not only to develop biotechnological tools and smart nanomaterials, but also to treat pathologies in new ways. This Review focuses on recent progress made in the exciting area pertaining the interaction between peptide assemblies and proteins. Earlier works aimed to identify proteins able to bind peptide assemblies for therapeutics’ delivery and biocompatible scaffolds. Recent advancements cover more applications that go beyond tissue regeneration and biomaterials. On one hand, self‐assembling peptides interacting with proteins can inhibit pathological amyloid fibrillation, or boost the immune response to vaccines. On the other, they have been exploited to promote protein crystallization, also for therapeutics’ delivery. As research advances in this exciting area, it opens the way towards a qualitative leap in the clinical translation of supramolecular medicinal chemistry to creatively tackle unmet challenges of modern disease treatment and prevention.

Template-Assisted Crystallization Behavior in Stirred Solutions of the Monoclonal Antibody Anti-CD20: Probability Distributions of Induction Times.

We present a method to determine the template crystallization behavior of proteins. This method is a statistical approach that accounts for the stochastic nature of nucleation. It makes use of batch-wise experiments under stirring conditions in volumes smaller than 0.3 mL to save material while mimicking larger-scale processes. To validate our method, it was applied to the crystallization of a monoclonal antibody of pharmaceutical interest, Anti-CD20. First, we determined the Anti-CD20 phase diagram in a PEG-400/Na2SO4/water system using the batch method, as, to date, no such data on Anti-CD20 solubility have been reported. Then, the probability distribution of induction times was determined experimentally, in the presence of various mesoporous silica template particles, and crystallization of Anti-CD20 in the absence of templates was compared to template-assisted crystallization. The probability distribution of induction times is shown to be a suitable method to determine the effect of template particles on protein crystallization. The induction time distribution allows for the determination of two key parameters of nucleation, the nucleation rate and the growth time. This study shows that the use of silica particles leads to faster crystallization and a higher nucleation rate. The template particle characteristics are shown to be critical parameters to efficiently promote protein crystallization.

Protein aggregation and crystallization with ionic liquids: Insights into the influence of solvent properties

Ionic liquids (ILs) have been used in solvents for proteins in many applications, including biotechnology, pharmaceutics, and medicine due to their tunable physicochemical and biological properties. Protein aggregation is often undesirable, and predominantly occurs during bioprocesses, while the aggregation process can be reversible or irreversible and the aggregates formed can be native/non-native and soluble/insoluble. Recent studies have clearly identified key properties of ILs and IL-water mixtures related to protein performance, suggesting the use of the tailorable properties of ILs to inhibit protein aggregation, to promote protein crystallization, and to control protein aggregation pathways. This review discusses the critical properties of IL and IL-water mixtures and presents the latest understanding of the protein aggregation pathways and the development of IL systems that affect or control the protein aggregation process. Through this feature article, we hope to inspire further advances in understanding and new approaches to controlling protein behavior to optimize bioprocesses.

Effects of Small Biomolecules on Lysozyme Crystallization

To develop appropriate biocompatible nucleants, glutathione (GSH), glycine (Gly) and cysteine (Cys) were used as the biomolecular additives to study their effect on the regulation of lysozyme crystallization. Characterized by Fourier transform infrared spectroscopy, powder X-ray diffraction, circular dichroism, etc., the results show that GSH can effectively promote protein crystallization under even lower concentrations of lysozyme by enhancing the nucleation rate, comparing with the additives of Gly and Cys, whereas lysozyme crystal structures produced in the presence of the biomolecular additives are similar to those in the absence of the additives. It shows in combination with molecular modeling that the stronger interactions between small biomolecular additives and the lysozyme LOOP accelerate the heterogeneous nucleation. It is suggested that such small biomolecules can be used as promising nucleants for promoting protein crystallization in the food and pharmaceutical industries.

DNA Origami as Seeds for Promoting Protein Crystallization.

This study reports the first experimental evidence of DNA origami as a seed resulting in the increase in probability of protein crystallization. Using the DNA origami constructed from long single-stranded M13 DNA scaffolds folded with short single-stranded DNA staples, it was found that the addition of the DNA origami in concentrations of 2-6 nM to mixtures of a well-characterized protein (catalase) solution (1.0-7.0 mg/mL) resulted in a higher proportion of mixtures with successful crystallization, up to 11× greater. The improvement in crystallization is evident particularly for mixtures with low concentrations of catalase (<5 mg/mL). DNA origami in different conformations of a flat rectangular sheet and a tubular hollow cylinder were examined. Both conformations improved the crystallization as compared to control experiments without M13 DNA or nonfolded M13 DNA but exhibited little difference in the extent of protein crystallization improvement. This work confirms the predictions of the potential use of DNA origami to promote protein crystallization, with potential application to systems with limited protein availability or difficulty in crystallization.

Effect of real-world sounds on protein crystallization

Protein crystallization is sensitive to the environment, while audible sound, as a physical and environmental factor during the entire process, is always ignored. We have previously reported that protein crystallization can be affected by a computer-generated monotonous sound with fixed frequency and amplitude. However, real-world sounds are not so simple but are complicated by parameters (frequency, amplitude, timbre, etc.) that vary over time. In this work, from three sound categories (music, speech, and environmental sound), we selected 26 different sounds and evaluated their effects on protein crystallization. The correlation between the sound parameters and the crystallization success rate was studied mathematically. The results showed that the real-world sounds, similar to the artificial monotonous sounds, could not only affect protein crystallization, but also improve crystal quality. Crystallization was dependent not only on the frequency, amplitude, volume, irradiation time, and overall energy of the sounds but also on their spectral characteristics. Based on these results, we suggest that intentionally applying environmental sound may be a simple and useful tool to promote protein crystallization.

Seeding Protein Crystallization with Cross-Linked Protein Crystals

Protein crystallization is of great importance because protein crystals have a number of different important applications, including large-scale purification of proteins, determination of protein structure, nanoparticle preparation, and theoretical studies of crystallization. An approach often used to efficiently crystallize proteins is the use of nucleants or seeds (small fragments of protein crystals) that can help increase the probability of protein crystallization. Due to the very positive effect that seeding has on protein crystallization, seeds are now widely accepted and utilized in practical protein crystallization. Here, we show that cross-linked protein crystals (CLPCs), which retain the crystal structure but are much more stable than non-cross-linked crystals, can also be used as a new type of seed for promoting protein crystallization. Seeding with CLPCs has effects on both the reproducibility and screening of protein crystals and could improve the optical perfection (well-defined facets) of protein crystals and the probability of obtaining protein crystals. In addition, the cross-linked protein crystals may reduce the concentration of protein molecules needed to obtain protein crystals. Furthermore, CLPCs are very stable in air, and no protective medium is necessary for the long-term storage of CLPCs. This feature makes the CLPC seeding method a potentially powerful technique in practical protein crystallization on either a laboratorial or an industrial scale.

Utilization of Cyclodextrins and Its Derivative Particles as Nucleants for Protein Crystallization

Finding new nucleants to promote protein crystallization is an important task, especially for purposes other than structural determination. Here, we investigated cyclodextrins and its derivative particles, as potential nucleants for protein crystallization. β-cyclodextrin (β-CD) and its derivatives (including p-toluenesulfonyl-β-cyclodextrin (PTCD), polymer-β-cyclodextrin (PCD), Mono-(6-(1,6-hexamethylenediamine)-6-deoxy)-β-Cyclodextrin (MHCD) and Mercapto-β-cyclodextrin (MCD)) were used as nucleants. The experimental results confirmed that β-CD and its derivatives showed significantly positive effects, promoting protein crystallization and improving crystal quality. A larger number of protein molecules (including lysozyme, catalase, subtilisin A VIII, concanavalin A VI, α-chymotrypsinogen, proteinase K, cellulase, papain, glucose isomerase, hemoglobin and ribonuclease A XII) attached to the particles usually corresponded to a higher crystallization success rate. More detailed analysis showed that cyclode...

An investigation on the effect of surface roughness of crystallization plate on protein crystallization

Surface treatment by oxidizing the crystallization plates can significantly promote protein crystallization and requires no change to routine screening protocols; therefore, it is potentially useful for practical protein crystallization screening. However, experiments have shown that the amount of oxidants and the treatment process need to be optimized to achieve effective results. Searching for the suitable amount of oxidants, temperature and processing time for surface treatment of the plate will increase the workload and decrease the efficiency of the screening process. To solve this problem, a series of trials to determine suitable surface treatment conditions were conducted. Based on these experiments, not only was the most suitable processing condition for the optimal protein crystallization screening hits discovered but also the relationship between the treatment process and the protein crystallization screening hits was explored. With these results, the surface treatment of protein crystallization plates became easier and more effective, allowing the oxidizing surface treatment method to be applied on plates for use in routine protein crystallization screening.

Generating Recombinant Antibodies to Membrane Proteins through Phage Display.

One of the most important classes of proteins in terms of drug targets is cell surface membrane proteins, and yet it is a challenging set of proteins for generating high-quality affinity reagents. In this review, we focus on the use of phage libraries, which display antibody fragments, for generating recombinant antibodies to membrane proteins. Such affinity reagents generally have high specificity and affinity for their targets. They have been used for cell staining, for promoting protein crystallization to solve three-dimensional structures, for diagnostics, and for treating diseases as therapeutics. We cover publications on this topic from the past 10 years, with a focus on the various formats of membrane proteins for affinity selection and the diverse affinity selection strategies used. Lastly, we discuss the challenges faced in this field and provide possible directions for future efforts.

Effect of Audible Sound on Protein Crystallization

The successful crystallization of proteins is important because their molecular three-dimensional structures can be obtained through X-ray diffraction when in their crystalline form. Investigation of the crystallization process is beneficial for this purpose. We have reported that protein crystallization is sensitive to audible sound, which is commonly present but is often ignored. Here we investigate the effect of audible sound parameters, especially frequency, on a protein crystallization. We show a significant facilitation of protein crystallization using 5000 Hz audible sound, possible mechanism was also tried to be clarified. Suitably controlled audible sound can be beneficial for promoting protein crystallization. Therefore, audible sound can be used as a simple tool to promote protein crystallization. In addition, the processing of other types of materials may also be affected by audible sound.

Promoting protein crystallization using a plate with simple geometry

Increasing the probability of obtaining protein crystals in crystallization screening is always an important goal for protein crystallography. In this paper, a new method called the cross-diffusion microbatch (CDM) method is presented, which aims to efficiently promote protein crystallization and increase the chance of obtaining protein crystals. In this method, a very simple crystallization plate was designed in which all crystallization droplets are in one sealed space, so that a variety of volatile components from one droplet can diffuse into any other droplet via vapour diffusion. Crystallization screening and reproducibility tests indicate that this method could be a potentially powerful technique in practical protein crystallization screening. It can help to obtain crystals with higher probability and at a lower cost, while using a simple and easy procedure.

Deep hydration: Poly(ethylene glycol) Mw 2000–8000 Da probed by vibrational spectrometry and small-angle neutron scattering and assignment of ΔG° to individual water layers

Aqueous solutions of poly(ethylene glycol) (PEG) exhibit some remarkable properties, among which is the small changes in water activity compared to the volumes occupied by the PEG: For example, the water in a 20% mass fraction solution of 6000 Da PEG has an activity of 0.9939. We have investigated PEGs with molecular weights 200, 400, 1000, 2000, 4000, and 8000 Da in the concentration range 1% to 17% mass fraction at neutral pH and with added KCl concentrations of 10 mmol L−1 in aqueous solutions–conditions near those for promoting protein crystallization. These solutions exhibit a structural change at around 6% mass fraction as seen in the solution viscosities, compressibilities, and infrared spectra. Raman spectroscopy shows that the PEGs remain in the same structural form over the concentration range, and the infrared spectra indicate that the change must be due to a local shift in the water structure. Modeling of the results from small-angle neutron scattering (SANS) on the solutions suggests that the structures of the PEGs in the molecular mass range 2000 Da to 8000 Da are paired in the solution, and the separation distance decreases with increasing PEG concentration. From the structure, it becomes clear that the small effect on water activity occurs because of screening by the more weakly bound outer layers. From the bulk measurement of aw and with reasonable assumptions, a free energy ΔG° can be assigned to each of the fourth, third, and second hydration layers.

Structure of Collagen Receptor Integrin α1I Domain Carrying the Activating Mutation E317A

We have analyzed the structure and function of the integrin α(1)I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α(1)I C139S/E317A was a higher avidity collagen binder than α(1)I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α(1)I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α(2)I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α(2)I structure, has not changed its position in the activated α(1)I variant. During the integrin activation, Glu(335) on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β(1) subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu(335). This indicates that the stabilization of helix 7 into its downward position is not required if the α(1) MIDAS is already open. To conclude, the activated α(1)I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg(287)-Glu(317) ion pair has just broken during the integrin activation.

Small temperature oscillations promote protein crystallization

Small temperature oscillations (TOs) are shown to increase the success rates of protein crystallization experiments by enhancing protein nucleation and by inducing the formation of larger crystals. This effect was initially found when we accidentally used a defective crystallization incubator and it is now confirmed for the crystallization of egg-white lysozyme at 14 and 20 °C, and also glucose isomerase at 20 °C, both under experimental conditions close to the nucleation zone of the proteins' phase diagram. Measurements of the nucleation times at constant lysozyme and precipitant concentrations show that the nucleation promoting effect is due to favourable crystallization pathways induced by TOs. The imposed TOs were ±1.0 °C during complete cycles of 20 min. X-Ray diffraction studies show worst and more disperse resolution limits when the lysozyme crystals are grown at constant temperature (2.05 ± 0.51 A without TOs, and 1.79 ± 0.15 A with TOs).

Potential use of ultrasound to promote protein crystallization

This work shows promising applications of ultrasound in promoting protein crystallization, which is important for structure determination by X-ray crystallography. It was observed that ultrasound can be used as a nucleation promoter as it decreases the energy barrier for crystal formation. Crystallization experiments on egg-white lysozyme were carried out with and without ultrasonic irradiation using commercial crystallization plates placed in temperature-controlled water baths. The nucleation-promoting effect introduced by ultrasound is illustrated by the reduction of the metastable zone width, as measured by the isothermal microbatch technique. The same effect was confirmed by the increased number of conditions leading to the formation of crystals when vapour diffusion techniques were carried out in the presence of ultrasound. By inducing faster nucleation, ultrasound leads to protein crystals grown at low supersaturation levels, which are known to have better diffraction properties. In fact, X-ray diffraction data sets collected using 13 lysozyme crystals (seven grown with ultrasound and six without) show an average 0.1 Å improvement in the resolution limit when ultrasound was used (p&lt; 0.10). Besides the immediate application of ultrasound in nucleation promotion, the preliminary diffraction results also suggest a promising application in crystal quality enhancement.

Application of high throughput ligand screening to promote protein crystallization

Application of high throughput ligand screening to promote protein crystallization