Attenuated total reflection-FT-IR spectroscopic imaging of protein crystallization

Protein crystallization is of strategic and commercial relevance in the post-genomic era because of its pivotal role in structural proteomics projects. Although protein structures are crucial for understanding the function of proteins and to the success of rational drug design and other biotechnology applications, obtaining high quality crystals is a major bottleneck to progress. The major means of obtaining crystals is by massive-scale screening of a target protein solution with numerous crystallizing agents. However, when crystals appear in these screens, one cannot easily know if they are crystals of protein, salt, or any other molecule that happens to be present in the trials. We present here a method based on Attenuated Total Reflection (ATR)-FT-IR imaging that reliably identifies protein crystals through a combination of chemical specificity and the visualizing capability of this approach, thus solving a major hurdle in protein crystallization. ATR-FT-IR imaging was successfully applied to study the crystallization of thaumatin and lysozyme in a high-throughput manner, simultaneously from six different solutions. This approach is fast as it studies protein crystallization in situ and provides an opportunity to examine many different samples under a range of conditions.

Purification and crystallization of the hydroxylase component of the methanesulfonate monooxygenase from Methylosulfonomonas methylovora strain M2

The aim of the work described in this paper was two-fold: (1) the purification of the hydroxylase component of the MSAMO to electrophoretic homogeneity using a four-step chromatographic strategy and (2) the crystallization of the two-component hydroxylase of the MSAMO in order to enhance our understanding of the precise three-dimensional structure of the MSAMO, thus yielding an insight into the nature of the active site of this enzyme. Optimised crystallization conditions were identified allowing growth of crystals of the hydroxylase component of the MSAMO within five days. Crystals exhibited a brown colour suggesting the presence on an intact Rieske-iron sulfur centre and diffracted to 7.0 Å when a few degrees of data were evaluated on a beam line X11. © 2006 Elsevier Inc. All rights reserved.

Optimization of protein crystallization:the OptiCryst project

Protein crystallization has gained a new strategic and commercial relevance in the postgenomic era due to its pivotal role in structural genomics. Producing high quality crystals has always been a bottleneck to efficient structure determination, and this problem is becoming increasingly acute. This is especially true for challenging, therapeutically important proteins that typically do not form suitable crystals. The OptiCryst consortium has focused on relieving this bottleneck by making a concerted effort to improve the crystallization techniques usually employed, designing new crystallization tools, and applying such developments to the optimization of target protein crystals. In particular, the focus has been on the novel application of dual polarization interferometry (DPI) to detect suitable nucleation; the application of in situ dynamic light scattering (DLS) to monitor and analyze the process of crystallization; the use of UV-fluorescence to differentiate protein crystals from salt; the design of novel nucleants and seeding technologies; and the development of kits for capillary counterdiffusion and crystal growth in gels. The consortium collectively handled 60 new target proteins that had not been crystallized previously. From these, we generated 39 crystals with improved diffraction properties. Fourteen of these 39 were only obtainable using OptiCryst methods. For the remaining 25, OptiCryst methods were used in combination with standard crystallization techniques. Eighteen structures have already been solved (30% success rate), with several more in the pipeline.

In situ studies of phase separation and crystallization directed by Marangoni instabilities during spin-coating

Results of a pioneering study are presented in which for the first time, crystallization, phase separation and Marangoni instabilities occurring during the spin-coating of polymer blends are directly visualized, in real-space and real-time. The results provide exciting new insights into the process of self-assembly, taking place during spin-coating, paving the way for the rational design of processing conditions, to allow desired morphologies to be obtained. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insights into the influence of solvent polarity on the crystallization of poly(ethylene oxide) spin-coated thin films via in situ grazing incidence wide-angle X-ray scattering

Controlling polymer thin-film morphology and crystallinity is crucial for a wide range of applications, particularly in thin-film organic electronic devices. In this work, the crystallization behavior of a model polymer, poly(ethylene oxide) (PEO), during spin-coating is studied. PEO films were spun-cast from solvents possessing different polarities (chloroform, THF, and methanol) and probed via in situ grazing incidence wide-angle X-ray scattering. The crystallization behavior was found to follow the solvent polarity order (where chloroform < THF < methanol) rather than the solubility order (where THF > chloroform > methanol). When spun-cast from nonpolar chloroform, crystallization largely followed Avrami kinetics, resulting in the formation of morphologies comprising large spherulites. PEO solutions cast from more polar solvents (THF and methanol) do not form well-defined highly crystalline morphologies and are largely amorphous with the presence of small crystalline regions. The difference in morphological development of PEO spun-cast from polar solvents is attributed to clustering phenomena that inhibit polymer crystallization. This work highlights the importance of considering individual components of polymer solubility, rather than simple total solubility, when designing processing routes for the generation of morphologies with optimum crystallinities or morphologies.

A hydrothermally stable transition alumina by condensation-enhanced self-assembly and pyrolysis crystallization:application in the steam reforming of methane

The preparation of a steam-based hydrothermally stable transition alumina is reported. The gel was derived from a synthetic sol-gel route where Al-tri-sec-butoxide is hydrolysed in the presence of a non-ionic surfactant (EO20PO70EO20), HCl as the catalyst and water (H2O/Al = 6); the condensation was enhanced by treating the hydrolysed gel with tetrabutylammonium hydroxide (TBAOH), after which it was dried at 60 °C by solvent evaporation. The so-obtained mesophase was crystallized under argon at 1200 °C (1 h) producing a transition alumina containing δ/α, and possibly θ, alumina phases. Due to its surface acidity, the pyrolysis conditions transform the block copolymer into a cross-linked char structure that embeds the alumina crystallites. Calcination at 650 °C generates a fully porous material by burning the char; a residual carbon of 0.2 wt.% was found, attributed to the formation of surface (oxy)carbides. As a result, this route produces a transition alumina formed by nanoparticles of about 30 nm in size on average, having surface areas in the range of 59-76 m2 g-1 with well-defined mesopores centered at 14 nm. The material withstands steam at 900 °C with a relative surface area rate loss lower than those reported for δ-aluminas, the state-of-the-art MSU-X γ-alumina and other pure γ-aluminas. The hydrothermal stability was confirmed under relevant CH4 steam reforming conditions after adding Ni; a much lower surface area decay and higher CH4 conversion compared to a state-of-the-art MSU-X based Ni catalyst were observed. Two effects are important in explaining the properties of such an alumina: the char protects the particles against sintering, however, the dominant effect is provided by the TBAOH treatment that makes the mesophase more resistant to coarsening and sintering. This journal is © the Partner Organisations 2014.