Crystallizing membrane proteins using lipidic mesophases

peer-reviewedThis paper was obtained through PEER (Publishing and the Ecology of European Research) http://www.peerproject.euA detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human engineered β2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour

The Effect of TBP-Related Factor 2 on Chromocenter Formation and Chromosome Segregation in Drosophila Melanogaster

Chromosome nondisjunction in meiosis causes the gene disbalance and a number of anomalies in development and fertility. Otherwise, genetically programmed sex-ratio meiotic drive occurs in a number of species. One of the forms of eukaryotic genome organization is a chromocenter evolutionally involved in the regulation of chromosome behavior in dividing cells among insects, plants, mammals, mollusks, and even yeast. In Drosophila, TBP related factor 2 (Trf2) belongs to a conservative Tbp (TATA box-binding protein) gene family and encodes a basic transcription factor. Recent data demonstrates that a decrease in TRF2 expression can result in the abnormalities of chromatin condensation; however, no details of this process have been studied. We demonstrated that a decrease in the TRF2 expression damaged proper chromocenter structure and abolished chromatin condensation and it was a reason for the chromosome nondisjunction. We found that compact chromocenter and correct homologue pairing were abolished in flies with a lower Trf2 expression in germline and in somatic cells. We conclude that TRF2 can not only be involved in transcription activation, but also may perform structural function in pericentromeric heterochromatin organization. The possibility of TRF2 to regulate the evolutionary genetically programmed sex-ratio meiotic drive is discussed

Modelling Convective Dissolution and Reaction of Carbon Dioxide in Saline Aquifers

In an effort to reduce atmospheric carbon dioxide (CO2) emissions and mitigate climate change, it has been proposed to sequester supercritical CO2 in underground saline aquifers. Geological storage of CO2 involves different trapping mechanisms which are not yet fully understood. In order to improve the understanding of the effect of chemical reaction on the flow and transport of CO2, these storage mechanisms are modelled experimentally and numerically in this work. In particular, the destabilising interaction between the fluid hydrodynamics and a density-increasing second-order chemical reaction is considered. It is shown that after nondimensional scaling, the flow in a given physicochemical system is governed by two dimensionless groups, Da/Ra2, which measures the timescale for convection compared to those for reaction and diffusion, and CBo', which reflects the excess of the environmental reactant species relative to the diffusing solute. The destabilising reactive scenario is modelled experimentally under standard laboratory conditions using an immiscible two-layer system with acetic acid acting as the solute. A novel colorimetric technique is developed to infer the concentrations of chemical species from the pH of the solution making it possible to measure the flux of solute into the aqueous domain. The validity of this experimental system as a suitable analogue for the dissolution of CO2 is tested against previous work and the destabilising effect of reaction is investigated by adding ammonia to the lower aqueous layer. The system is also modelled numerically and it is shown that the aqueous phase reaction between acetic acid and ammonia can be considered to be instantaneous, meaning that Da/Ra2 tends to infinity and the flow is therefore governed only by the initial dimensionless concentration of reactant in the aqueous phase. The results from the experiments and numerical simulations are in good agreement, showing that an increase in the initial concentration of reactant increases the destabilising effect of reaction, accelerates the onset of convection and enhances the rate of dissolution of solute. The numerical model is then applied to a real world aquifer in the Sleipner gas field and it is demonstrated how the storage capacity of a potential CO2 reservoir could be enhanced by chemical reactionEPSR

Structural basis for bifunctional peptide recognition at human δ-opioid receptor

Bi-functional μ- and δ- opioid receptor (OR) ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects. We solved the structure of human δ-OR bound to the bi-functional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt(1)-Tic(2)-Phe(3)-Phe(4)-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt(1) and Tic(2). The observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides, and to develop improved analgesics

Monoolein Lipid Phases as Incorporation and Enrichment Materials for Membrane Protein Crystallization

The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase

Xenobiotic-induced activation of human aryl hydrocarbon receptor target genes in Drosophila is mediated by the epigenetic chromatin modifiers

Aryl hydrocarbon receptor (AHR) is the key transcription factor that controls animal development and various adaptive processes. The AHR\u27s target genes are involved in biodegradation of endogenous and exogenous toxins, regulation of immune response, organogenesis, and neurogenesis. Ligand binding is important for the activation of the AHR signaling pathway. Invertebrate AHR homologs are activated by endogenous ligands whereas vertebrate AHR can be activated by both endogenous and exogenous ligands (xenobiotics). Several studies using mammalian cultured cells have demonstrated that transcription of the AHR target genes can be activated by exogenous AHR ligands, but little is known about the effects of AHR in a living organism. Here, we examined the effects of human AHR and its ligands using transgenic Drosophila lines with an inducible human AhR gene. We found that exogenous AHR ligands can increase as well as decrease the transcription levels of the AHR target genes, including genes that control proliferation, motility, polarization, and programmed cell death. This suggests that AHR activation may affect the expression of gene networks that could be critical for cancer progression and metastasis. Importantly, we found that AHR target genes are also controlled by the enzymes that modify chromatin structure, in particular components of the epigenetic Polycomb Repressive complexes 1 and 2. Since exogenous AHR ligands (alternatively - xenobiotics) and small molecule inhibitors of epigenetic modifiers are often used as pharmaceutical anticancer drugs, our findings may have significant implications in designing new combinations of therapeutic treatments for oncological diseases. © Akishina et al

Radiomics of Lung Nodules: A Multi-Institutional Study of Robustness and Agreement of Quantitative Imaging Features.

Radiomics is to provide quantitative descriptors of normal and abnormal tissues during classification and prediction tasks in radiology and oncology. Quantitative Imaging Network members are developing radiomic "feature" sets to characterize tumors, in general, the size, shape, texture, intensity, margin, and other aspects of the imaging features of nodules and lesions. Efforts are ongoing for developing an ontology to describe radiomic features for lung nodules, with the main classes consisting of size, local and global shape descriptors, margin, intensity, and texture-based features, which are based on wavelets, Laplacian of Gaussians, Law's features, gray-level co-occurrence matrices, and run-length features. The purpose of this study is to investigate the sensitivity of quantitative descriptors of pulmonary nodules to segmentations and to illustrate comparisons across different feature types and features computed by different implementations of feature extraction algorithms. We calculated the concordance correlation coefficients of the features as a measure of their stability with the underlying segmentation; 68% of the 830 features in this study had a concordance CC of ≥0.75. Pairwise correlation coefficients between pairs of features were used to uncover associations between features, particularly as measured by different participants. A graphical model approach was used to enumerate the number of uncorrelated feature groups at given thresholds of correlation. At a threshold of 0.75 and 0.95, there were 75 and 246 subgroups, respectively, providing a measure for the features' redundancy

The Membrane Protein Data Bank

The Membrane Protein Data Bank (MPDB) is an online, searchable, relational database of structural and functional information on integral, anchored and peripheral membrane proteins and peptides. Data originates from the Protein Data Bank and other databases, and from the literature. Structures are based on X-ray and electron diffraction, nuclear magnetic resonance and cryoelectron microscopy. The MPDB is searchable online by protein characteristic, structure determination method, crystallization technique, detergent, temperature, pH, author, etc. Record entries are hyperlinked to the PDB and Pfam for viewing sequence, three-dimensional structure and domain architecture, and for downloading coordinates. Links to PubMed are also provided. The MPDB is updated weekly in parallel with the Protein Data Bank. Statistical analysis of MPDB records can be performed and viewed online. A summary of the statistics as applied to entries in the MPDB is presented. The data suggest conditions appropriate for crystallization trials with novel membrane proteins