Bimetallic MOF-Derived Solar-Triggered Monolithic Adsorbent for Enhanced Atmospheric Water Harvesting.

The development of economical, energy-saving, and efficient metal-organic framework (MOF)-based adsorbents for atmospheric water collection is highly imperative for the rapid advancement of renewable freshwater resource exploitation. Herein, a feasible one-step solvothermal formation strategy of bimetallic MOF (BMOF) is proposed and applied to construct a solar-triggered monolithic adsorbent for enhanced atmospheric water collection. Benefiting from the reorganization and adjustment of topology structure by Al atoms and Fe atoms, the resultant BMOF(3) consisting of Al-fumarate and MIL-88A has a higher specific surface area (1202.99 m2  g-1 ) and pore volume (0.51 cm3  g-1 ), thereby outperforming the parental MOFs and other potential MOFs in absorbing water. Expanding upon this finding, the solar-triggered monolithic adsorbent is further developed through a bottom-up assembly of polyaniline/chitosan layers and hybridized BMOF(3) skeletons on a glass fiber support. The resultant monolithic adsorbent exhibits superior sorption-desorption kinetics, leading to directional water transport and rapid solar-assisted vapor diffusion. As a proof-of-concept demonstration, an exquisite water harvester is constructed to emphasize a high water yield of 1.19 g g-1 per day of the designed monolithic adsorbent. Therefore, the design and validation of bimetallic MOF-derived solar-triggered adsorbent in this work are expected to provide a reference for the large-scale applications of MOF-based atmospheric water harvesting.

Current trends in protein crystallization.

UNLABELLED: Proteins belong to the most complex colloidal system in terms of their physicochemical properties, size and conformational-flexibility. This complexity contributes to their great sensitivity to any external change and dictate the uncertainty of crystallization. The need of 3D models to understand their functionality and interaction mechanisms with other neighbouring (macro)molecules has driven the tremendous effort put into the field of crystallography that has also permeated other fields trying to shed some light into reluctant-to-crystallize proteins. This review is aimed at revising protein crystallization from a regular-laboratory point of view. It is also devoted to highlight the latest developments and achievements to produce, identify and deliver high-quality protein crystals for XFEL, Micro-ED or neutron diffraction. The low likelihood of protein crystallization is rationalized by considering the intrinsic polypeptide nature (folded state, surface charge, etc) followed by a description of the standard crystallization methods (batch, vapour diffusion and counter-diffusion), including high throughput advances. Other methodologies aimed at determining protein features in solution (NMR, SAS, DLS) or to gather structural information from single particles such as Cryo-EM are also discussed. Finally, current approaches showing the convergence of different structural biology techniques and the cross-methodologies adaptation to tackle the most difficult problems, are presented. SYNOPSIS: Current advances in biomacromolecules crystallization, from nano crystals for XFEL and Micro-ED to large crystals for neutron diffraction, are covered with special emphasis in methodologies applicable at laboratory scale.

Studies on Aspirin Crystals Generated by a Modified Vapor Diffusion Method.

The objectives of the current investigation were (1) to study the influence of selected two different non-solvents (diethylether and dichloromethane) on the drug crystal formation of a model drug, aspirin (ASP-I) by the modified vapor diffusion method and (2) to characterize and compare the generated crystals (ASP-II and ASP-III) using different analytical techniques with that of unprocessed ASP-I. When compared to the classical vapor diffusion method which consumes about 15 days to generate drug crystals, the modified method needs only 12 h to get the same. Fourier transform-infrared spectroscopy (FT-IR) reveals that the internal structures of ASP-II and ASP-III crystals were identical when compared with ASP-I. Although the drug crystals showed a close similarity in X-ray diffraction patterns, the difference in the relative intensities of some of the diffraction peaks (especially at 2θ values of around 7.7 and 15.5) could be attributed to the crystal habit or crystal size modification. Similarly, the differential scanning calorimetry (DSC) study speculates that only the crystal habit modifications might occur but without involving any change in internal structure of the generated drug polymorphic form I. This is further substantiated from the scanning electron microscopy (SEM) pictures that indicated the formation of platy shape for the ASP-II crystals and needle shape for the ASP-III crystals. In addition, the observed slow dissolution of ASP crystals should indicate polymorph form I formation. Thus, the modified vapor diffusion method could routinely be used to screen and legally secure all possible forms of other drug entities too.

Efficient cryoprotection of macromolecular crystals using vapor diffusion of volatile alcohols.

Macromolecular X-ray crystallography, usually done at cryogenic temperature to limit radiation damage, often requires liquid cryoprotective soaking that can be labor intensive and damaging to crystals. Here we describe a method for cryoprotection that uses vapor diffusion of volatile cryoprotective agents into loop-mounted crystals. The crystal is mounted into a vial containing a small volume of an alcohol-based cryosolution. After a short incubation with the looped crystal sitting in the cryosolution vapor, the crystal is transferred directly from the vial into the cooling medium. Effective for several different protein crystals, the approach obviates the need for liquid soaking and opens up a heretofore underutilized class of cryoprotective agents for macromolecular crystallography.

How to grow crystals for X-ray crystallography.

Growing high-quality crystals remains a necessary part of crystallography and many other techniques. This article tabulates and describes several techniques and variations that will help individuals grow high-quality crystals in preparation for crystallographic techniques and other endeavors, such as form screening. The discussion is organized to focus on low-tech approaches available in any laboratory.

Apatite-coated outer layer eggshell membrane: A novel osteoinductive biohybrid composite for guided bone/tissue regeneration.

Hybrid biomimetic materials aim to replicate the organic-inorganic constructs of mineralized tissues. During eggshell formation, the outer surface of the eggshell membrane (ESM) promotes calcium carbonate nucleation, while the inner one prevents mineralization toward the egg white and yolk. In the current study, the outer surface of the ESM acted as a heteronucleant in calcium phosphate precipitation by the vapor diffusion sitting drop method, while the inner one remained unmineralized. The aim was to fabricate a 2D biomaterial with dual functions, osteoinductive on one side and protective against cell invasion on the other side. The microstructural, physicochemical, morphological, and mechanical properties of the mineralized ESM were characterized by XRD, TGA, XPS, FTIR/Raman, HR-SEM, and mechanical testing techniques. The cytocompatibility and osteoinductive ability were assessed by biological assays of cell viability, proliferation, and osteogenic differentiation on human mesenchymal stromal cells (hMSCs). Results indicate that the outer surface of the ESM induces the heterogeneous precipitation of carbonate-apatite phase depicting biomimetic features. In addition, the apatite/ESM shows a much higher cytocompatibility than the pristine ESM and promotes the osteogenic differentiation of hMSCs more efficiently. Overall, the apatite/ESM composite exhibits compositional, crystalline, mechanical, and biological properties that resemble those of mineralized tissues, rendering it an approachable and novel material especially useful in guided tissue/bone regeneration.

Spatially Confined Face-Selective Growth of Large-Area 2D Organic Molecular Crystals in a Supramolecular Gel for Highly Efficient Flexible Photodetection.

2D organic molecular crystals (2DOMCs) are promising materials for the fabrication of high-performance optoelectronic devices. However, the growth of organic molecules into 2DOMCs remains a challenge because of the difficulties in controlling their self-assembly with a preferential orientation in solution-process crystallization. Herein, fullerene is chosen as a model molecule to develop a supramolecular gel crystallization approach to grow large-area 2DOMCs by controlling the perfect arrangement on the {220} crystal plane with the assistance of a gelated solvent. In this case, the gel networks provide tuneable confined spaces to control the crystallization kinetics toward the growth of dominant crystal faces by their inhibiting motions of solvent or solute molecules to enable the growth of perfect crystals at appropriate nucleation rates. As a result, a large-area fullerene 2DOMC is produced successfully and its corresponding device on a flexible substrate exhibits excellent bendable properties and ultra-high weak light detection ability (2.9 × 1011 Jones) at a 10 V bias upon irradiation with 450 nm incident light. Moreover, its photoelectric properties remain unchanged after 200 cycles of bending at angles of 45, 90, and 180°. These results can be extended to the growth of other 2DOMCs for potentially fabricating advanced organic (opto)electronics.

Protein Crystallization of Two Recombinant Lpt Proteins.

The prerequisite for 3D structure determination of macromolecules via X-ray crystallography is well-ordered, diffracting crystals. Here, we report the recombinant production, biophysical/biochemical protein sample characterization, and vapor diffusion sitting drop crystallization protocols for two lipopolysaccharide transport proteins: LptH from Pseudomonas aeruginosa (Pa-LptH) and an inactive LptC mutant (G153R) from Escherichia coli (EcLptC24-191G153R).

Large crystal growth for neutron protein crystallography.

The use of neutron protein crystallography (NPX) is expanding rapidly, with most structures determined in the last decade. This growth is stimulated by a number of developments, spanning from the building of new NPX beamlines to the availability of improved software for structure refinement. The main bottleneck preventing structural biologists from adding NPX to the suite of methods commonly used is the large volume of the individual crystals required for a successful experiment. A survey of deposited NPX structures in the Protein Data Bank shows that about two-thirds came from crystals prepared using vapor diffusion, while batch and dialysis-based methods all-together contribute to most of the remaining one-third. This chapter explains the underlying principles of these protein crystallization methods and provides practical examples that may help others to successfully prepare large crystals for NPX.

Successful amphiphiles as the key to crystallization of membrane proteins: Bridging theory and practice.

BACKGROUND: Membrane proteins constitute a major group of proteins and are of great significance as pharmaceutical targets, but underrepresented in the Protein Data Bank. Particular reasons are their low expression yields and the constant need for cautious and diligent handling in a sufficiently stable hydrophobic environment substituting for the native membrane. When it comes to protein crystallization, such an environment is often established by detergents. SCOPE OF REVIEW: In this review, 475 unique membrane protein X-ray structures from the online data bank "Membrane proteins of known 3D structure" are presented with a focus on the detergents essential for protein crystallization. By systematic analysis of the most successful compounds, including current trends in amphiphile development, we provide general insights for selection and design of detergents for membrane protein crystallization. MAJOR CONCLUSIONS: The most successful detergents share common features, giving rise to favorable protein interactions. The hydrophile-lipophile balance concept of well-balanced hydrophilic and hydrophobic detergent portions is still the key to successful protein crystallization. Although a single detergent compound is sufficient in most cases, sometimes a suitable mixture of detergents has to be found to alter the resulting protein-detergent complex. Protein crystals with a high diffraction limit involve a tight crystal packing generally favored by detergents with shorter alkyl chains. GENERAL SIGNIFICANCE: The formation of well-diffracting membrane protein crystals strongly depends on suitable surfactants, usually screened in numerous crystallization trials. The here-presented findings provide basic criteria for the assessment of surfactants within the vast space of potential crystallization conditions for membrane proteins.

Investigation of the confinement effect on the evaporation behavior of a droplet pinned on a micropillar structure.

Evaporation of sessile droplet suffers from reduced evaporation rate due to the confinement of vapor diffusion imposed by the bottom substrate. However, it is possible to change the evaporation behavior of a droplet by suspending it from the bottom substrate, in particular, supporting the droplet on a micropillar. This is expected to enable diffusion transport in the downward direction that will subsequently enhance evaporative transport. In this study, we investigate the diffusion confinement effect imposed by the bottom substrate and the side wall of the micropillar through numerical simulations and experimental investigation. The approximate solutions for total evaporation rate and local evaporative flux were subsequently derived from the total evaporation rate predicted by the simulation results. The simulation results, agreeing within 5% with the experimental measurements, show that increasing the micropillar height enhances the total evaporation rate from the suspended hemispherical droplet. This enhancement is due to a dramatic improvement of the local evaporation rate near the contact line region as micropillar heights increase. The micropillar heights examined for maximum evaporation rates were observed under substrate temperatures from 60-98 °C. The increasing pillar height leads to smaller vapor diffusion resistance but greater conduction resistance.

On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies.

Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, 'naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

Crystallographic Analysis of the CusBA Heavy-Metal Efflux Complex of Escherichia coli.

Crystallization is one of the most successful techniques used to determine protein structure, especially for membrane proteins. However, the application of this technique is not straightforward and often hampered by the difficulties associated with expression, purification, and crystallization. Here we present our protocol and methodology for crystallizing the CusBA adaptor-transporter complex of Escherichia coli. Using these procedures, we were able to produce the first co-crystal structure of a resistance-nodulation-cell division (RND) transporter in complex with its associated membrane fusion protein.

Protein Crystallization.

Protein crystallization was discovered by chance nearly 200 years ago and was developed in the late nineteenth century as a powerful purification tool, and a demonstration of chemical purity. The crystallization of proteins, nucleic acids, and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule, but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by manipulation of various parameters that include temperature, ionic strength, and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors, or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch, and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years due to the advent of practical, easy-to-use screening kits, and the application of laboratory robotics.

Antibacterial Activity of Cinnamaldehyde and Estragole Extracted from Plant Essential Oils against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit.

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker disease in kiwifruit. Antibacterial activity of plant essential oils (PEOs) originating from 49 plant species were tested against Psa by a vapor diffusion and a liquid culture assays. The five PEOs from Pimenta racemosa, P. dioica, Melaleuca linariifolia, M. cajuputii, and Cinnamomum cassia efficiently inhibited Psa growth by either assays. Among their major components, estragole, eugenol, and methyl eugenol showed significant antibacterial activity by only the liquid culture assay, while cinnamaldehyde exhibited antibacterial activity by both assays. The minimum inhibitory concentrations (MICs) of estragole and cinnamaldehyde by the liquid culture assay were 1,250 and 2,500 ppm, respectively. The MIC of cinnamaldehyde by the vapor diffusion assay was 5,000 ppm. Based on the formation of clear zones or the decrease of optical density caused by these compounds, they might kill the bacterial cells and this feature might be useful for managing the bacterial canker disease in kiwifruit.

Crystallization of membrane proteins by vapor diffusion.

X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high-quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization.

A simple technique to reduce evaporation of crystallization droplets by using plate lids with apertures for adding liquids.

A method is described for using plate lids to reduce evaporation in low-volume vapor-diffusion crystallization experiments. The plate lids contain apertures through which the protein and precipitants were added to different crystallization microplates (the reservoir was filled before fitting the lids). Plate lids were designed for each of these commonly used crystallization microplates. This system minimizes the dehydration of crystallization droplets containing just a few nanolitres of protein and precipitant, and results in more reproducible diffraction from the crystals. For each lid design, changes in the weight of the plates were used to deduce the rate of evaporation under different conditions of temperature, air movement, droplet size and precipitant. For comparison, the state of dehydration was also visually assessed throughout the experiment. Finally, X-ray diffraction methods were used to compare the diffraction of protein crystals that were conventionally prepared against those that were prepared on plates with plate lids. The measurements revealed that the plate lids reduced the rate of evaporation by 63-82%. Crystals grown in 5 nl drops that were set up with plate lids diffracted to higher resolution than similar crystals from drops that were set up without plate lids. The results demonstrate that plate lids can be instrumental for improving few-nanolitre crystallizations.

An electrically assisted device for protein crystallization in a vapor-diffusion setup.

A new easy-to-use device has been designed and implemented for electric field-induced protein crystallization in a vapor-diffusion configuration. The device not only controls crystal nucleation by means of the electrical current, but also favors crystal growth owing to its vapor-diffusion setup. Crystallization was conducted in the presence of an internal electric field and direct current. The proteins investigated were lysozyme, as model protein, and 2TEL-lysozyme (a synthetic protein consisting of two tandem alpha helix motifs connected to a lysozyme moiety). Lysozyme crystals that grew attached to the cathode were larger than those grown attached to the anode or in the absence of an electric current. On the other hand, crystals of 2TEL-lysozyme qualitatively showed a better X-ray diffraction pattern when grown in the presence of an electric current.