Molecular BioSystems

doi: 10.1039/c3mb90037apmid: N/A

doi: 10.1039/c3mb90039hpmid: N/A

Tanramluk, Duangrudee; Akavipat, Ruj; Charoensawan, Varodom

doi: 10.1039/c3mb70361dpmid: 24126492

Technological advances in crystallography have led to the ever-rapidly increasing number of biomolecular structures deposited in public repertoires. This undoubtedly shifts the bottleneck of structural biology research from obtaining high-quality structures to data analysis and interpretation. The recently available glasses-free autostereoscopic laptop offers an unprecedented opportunity to visualize and study 3D structures using a much more affordable, and for the first time, portable device. Together with a gamepad re-programmed for 3D structure controlling, we describe how the gaming technologies can deliver the output 3D images for high-quality viewing, comparable to that of a passive stereoscopic system, and can give the user more control and flexibility than the conventional controlling setup using only a mouse and a keyboard.

Niu, Wei; Guo, Jiantao

doi: 10.1039/c3mb70204apmid: 24080788

Fluorescent proteins are essential tools in biological research, ranging from the study of individual biological components to the interrogation of complex cellular systems. Fluorescent protein derived biosensors are increasingly applied to the study of biological molecules and events in living cells. The present review focuses on a specific class of fluorescent protein biosensors in which a genetically installed unnatural amino acid (UAA*) acts as the sensing element. Upon direct interaction with the analyte of interest, the chemical and/or physical properties of UAA* are altered, which triggers fluorescence property changes of the biosensor and generates readouts. In comparison to mutagenesis approaches within the standard genetic code, introduction of UAA*s with a unique functionality and chemical reactivity could broaden the scope of analytes and improve the specificity of biosensors. Nonconventional functional groups in fluorescent proteins enable sensor designs that are not readily accessible using the common twenty amino acids. Recent reports of UAA*-containing fluorescent protein sensors serve as excellent examples for the utility of such sensor design. We envisage that the integration of the two powerful chemical biology tools, fluorescent protein sensors and genetic incorporation of UAA*s, will lead to novel biosensors that can expand and deepen current understanding of cellular processes.

Mosca, Ettore; Milanesi, Luciano

doi: 10.1039/c3mb70327dpmid: 24121459

Nowadays, computational and statistical methods focusing on integrated analysis of omics data are necessary. A few approaches have been recently described in the literature and a small number of software packages are available. We have developed a new method to generate networks of biological components that incorporate multi-omics information. The novelty of this method relies on using a multi-objective (MO) optimization procedure in order to drive the identification of networks that are enriched according to several statistical estimators. The network-based analysis of omics with MO optimization described in this work can be applied to different types of omics and biological interactions. By using this approach we found protein networks that participate in the establishment of the increased basal differentiation observed in breast tumors of BRCA1-mutation carriers. Additionally, we showed how MO optimization can be used to carry out a network-based comparison among several omic data sets: using transcriptomic data from two types of breast tumors and the corresponding epithelial cells from which tumors were generated, we found a protein network that shows a strong and coherent (the same direction) differential expression when comparing each tumor with its respective epithelial tissue. We have also compared the transcriptional variation detected in three different types of tumors originated in breast, colon and pancreas with the corresponding healthy tissues. Despite the global low correlation observed in the three pairs of tumors, we found more similar networks regulated in the same direction in colon and pancreas tumor cells. In conclusion, we propose the network-based analysis of omics with MO optimization as a valid tool for integrated analysis of omics data.

Ramsubramaniam, Nikhil; Tao, Feng; Li, Shuwei; Marten, Mark R.

doi: 10.1039/c3mb70358dpmid: 24129742

We describe the use of an isobaric tagging reagent, Deuterium isobaric Amine Reactive Tag (DiART), for quantitative phosphoproteomic experiments. Using DiART tagged custom mixtures of two phosphorylated peptides from alpha casein and their non-phosphorylated counterparts, we demonstrate the compatibility of DiART with TiO2 affinity purification of phosphorylated peptides. Comparison of theoretical vs. experimental reporter ion ratios reveals accurate quantification of phosphorylated peptides over a dynamic range of more than 15-fold. Using DiART labelling and TiO2 enrichment (DiART-TiO2) with large quantities of proteins (8 mg) from the cell lysate of model fungus Aspergillus nidulans, we quantified 744 unique phosphopeptides. Overlap of median values of TiO2 enriched phosphopeptides with theoretical values indicates accurate trends. Altogether these findings confirm the feasibility of performing quantitative phosphoproteomic experiments in a cost-effective manner using isobaric tagging reagents, DiART.

Fukunaga, Keisuke; Hatanaka, Takaaki; Ito, Yuji; Taki, Masumi

doi: 10.1039/c3mb70379gpmid: 24072138

The site-specific introduction of a haloacetamide derivative into a designated cysteine on a displaying peptide on a capsid protein (gp10) of bacteriophage T7 has been achieved. This easiest gp10-based thioetherification (10BASEd-T) is carried out in one-pot without side reactions or loss of phage infectivity.

Anderson, Lindsey N.; Culley, David E.; Hofstad, Beth A.; Chauvigné-Hines, Lacie M.; Zink, Erika M.; Purvine, Samuel O.; Smith, Richard D.; Callister, Stephen J.; Magnuson, Jon M.; Wright, Aaron T.

doi: 10.1039/c3mb70333apmid:

Han, Junyan; Han, Myung Shin; Tung, Ching-Hsuan

doi: 10.1039/c3mb70269cpmid: 24056749

A cell permeable fluorescence turn-on probe, AcGQCy7, was developed to image β-galactosidase activity in living cells. Once internalized by β-galactosidase-expressing cells, the probe was hydrolyzed into a highly fluorescent molecule, and the fluorescent signal was retained in mitochondria for several days. This resulted in a long-lasting and strong β-galactosidase-dependent intracellular fluorescent signal with little background fluorescence in the culture media.

Andrews, Allison-Lynn; Nordgren, Ida Karin; Campbell-Harding, Gemma; Holloway, John W.; Holgate, Stephen T.; Davies, Donna E.; Tavassoli, Ali

doi: 10.1039/c3mb70298gpmid: 24056919

Interleukin-4 (IL-4) and Interleukin-13 (IL-13), key cytokines in the pathogenesis of allergic inflammatory disease, mediate their effects via a receptor composed of IL-13Rα1 and IL-4Rα. A third (decoy) receptor called IL-13Rα2 regulates interleukin signaling through this receptor complex. We employed a variety of biophysical and cell-based techniques to decipher the role of this decoy receptor in mediating IL-4 signaling though the IL-4Rα–IL-13Rα1 receptor complex. Surface plasmon resonance (SPR) analysis showed that IL-13Rα2 does not bind IL-4, and does not affect binding of IL-4 to IL-4Rα. These results indicate that the extracellular domains of IL-4Rα and IL-13Rα2 are not involved in the regulation of IL-4 signaling by IL-13Rα2. We next used a two-hybrid system to show that the cytoplasmic domains of IL-4Rα and IL-13Rα2 interact, and that the secondary structure of the IL-13Rα2 intracellular domain is critical for this interaction. The cellular relevance of this interaction was next investigated. BEAS-2B bronchial epithelial cells that stably express full length IL-13Rα2, or IL-13Rα2 lacking its cytoplasmic domain, were established. Over expression of IL-13Rα2 attenuated IL-4 and IL-13 mediated STAT6 phosphorylation. IL-13Rα2 lacking its cytoplasmic domain continued to attenuate IL-13-mediated signaling, but had no effect on IL-4-mediated STAT6 signaling. Our results suggest that the physical interaction between the cytoplasmic domains of IL-13Rα2 and IL-4Rα regulates IL-4 signaling through the IL-4Rα–IL-13Rα1 receptor complex.