The 2β Splice Variation Alters the Structure and Function of the Stromal Interaction Molecule Coiled-Coil Domains

Stromal interaction molecule (STIM)-1 and -2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels after oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil (CC) domains couple to PM Orai1 proteins and gate these Ca2+ release-activated Ca2+ (CRAC) channels, which facilitate store-operated Ca2+ entry (SOCE). Unlike STIM1 and STIM2, which are SOCE activators, the STIM2β splice variant contains an 8-residue insert located within the conserved CCs which inhibits SOCE. It remains unclear if the 2β insert further depotentiates weak STIM2 coupling to Orai1 or independently causes structural perturbations which prevent SOCE. Here, we use far-UV circular dichroism, light scattering, exposed hydrophobicity analysis, solution small angle X-ray scattering, and a chimeric STIM1/STIM2β functional assessment to provide insights into the molecular mechanism by which the 2β insert precludes SOCE activation. We find that the 2β insert reduces the overall α-helicity and enhances the exposed hydrophobicity of the STIM2 CC domains in the absence of a global conformational change. Remarkably, incorporation of the 2β insert into the STIM1 context not only affects the secondary structure and hydrophobicity as observed for STIM2, but also eliminates the more robust SOCE response mediated by STIM1. Collectively, our data show that the 2β insert directly precludes Orai1 channel activation by inducing structural perturbations in the STIM CC region.

Aging analysis of high voltage silicone rubber/silica nanocomposites under accelerated weathering conditions

Silicone rubber (SiR) is extensively used in outdoor insulation and other applications. However, like other polymers, SiR also degrades and lessens its performance in the exposition of environmental stresses. Nanofillers improve the aging behavior of polymeric insulating materials. To investigate the effect of nanosilica on the aging behavior of SiR, we fabricated nanocomposites of SiR with 2.5 wt.% nanosilica (SNC-2.5) and 5 wt.% nanosilica (SNC-5) by mechanical mixing and ultrasonication method. The prepared samples were subjected to uniform ultraviolet (UV) radiation, heat, humidity, salt fog, and acid rain along with neat SiR in cyclic manner at 2.5 kV for 5000 h in a specially fabricated weathering chamber. Neat SiR and both nanocomposites showed gradual decrease in transparency. Random loss and recovery in hydrophobicity and increase and decrease in leakage current (LC) were recorded for all samples, in which SNC-5 showed the best hydrophobic behavior and least LC. For SNC-5, Fourier transform infrared (FTIR) results showed negligible reduction in absorption peaks at important wave-numbers and increase and intactness were observed at absorption peaks related to the hydrophobic methyl group. Scanning electron microscopy (SEM) results also concurred with FTIR, LC measurements, and hydrophobicity analysis.

Hydrophobicity Analysis of Protein Primary Structures to Identify Helical Regions

Summary Objectives: A wavelet based approach for the hydrophobicity analysis of protein primary structures is proposed to predict the presence of alpha helices in the secondary structure. Methods: The information about hydropathy profile periodicity content together with a score of probability of occurrence of a single amino acid allows the localization of alpha helices. Results: The accuracy is comparable to other consolidated predictors based on different techniques (i.e.: neural networks, hidden markov models). Conclusion: This method is particularly suitable to capture the amphiphilic character of the helical structures.

EmrE, the smallest ion-coupled transporter, provides a unique paradigm for structure-function studies.

EmrE is an Escherichia coli multidrug transporter which confers resistance to a wide variety of toxicants by actively removing them in exchange for hydrogen ions. EmrE is a highly hydrophobic 12 kDa protein which has been purified by taking advantage of its unique solubility in organic solvents. After solubilization and purification, the protein retains its ability to transport as judged from the fact that it can be reconstituted in a functional form. Hydrophobicity analysis of the sequence yielded four putative transmembrane domains of similar sizes. Results from transmission Fourier transform infrared measurements agree remarkably well with this hypothesis and yielded alpha-helical estimates of 78% and 80% for EmrE in CHCl3:MeOH and 1,2-dimyristoyl phosphocholine, respectively. Furthermore, the fact that most of the amide groups in the protein do not undergo amide-proton H/D exchange implies that most (approximately 80%) of the residues are embedded in the bilayer. These observations are only consistent with four transmembrane helices. A domain lined by Cys41 and Cys95 accessible only to substrates such as the organic mercurial 4-(chloromercuri)benzoic acid has been identified. Both residues are asymmetric in their location with respect to the plane of the membrane, Cys95 being closer than Cys41 to the outside face of the membrane. In co-reconstitution experiments of wild-type protein with three different inactive mutants, negative dominance has been observed. This phenomenon suggests that EmrE is functional as a homo-oligomer.