The molecular basis of conformational instability of the ecdysone receptor DNA binding domain studied by in silico and in vitro experiments

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of our study was an elucidation of the molecular basis of this intriguing instability. Toward this end, we mapped the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the α-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. Our results might serve as a benchmark for further studies of the intricate nature of the EcR molecule

Dual FRET assay for detecting receptor protein interaction with DNA

We present here a new assay that is based on the idea of the molecular beacon. This assay makes it possible to investigate two proteins interacting with DNA at two binding sites that are close to each other. The effectiveness of the test depends on the exclusive binding of three DNA fragments in the presence of two proteins, and the monitoring of the process depends upon observing the quenching of two independent fluorescence donors. As a model we used the components of the heterodimeric ecdysteroid receptor proteins ultraspiracle (Usp) and ecdysone receptor (EcR) from Drosophila melanogaster and a response element from the promoter of the hsp27 gene. The response element consists of two binding sites (half-sites) for the DNA binding domains (DBDs). We have shown that protein–protein interactions mediate cooperative binding of the ecdysteroid receptor DBDs to a hsp27pal response element. The analysis of the microscopic dissociation constants obtained with the DMB led to the conclusion that there was increased affinity of UspDBD to the 5′ half-site in the presence of EcRDBD when the 3′ half-site was occupied, and increased affinity of EcRDBD to the 3′ half-site when the 5′ half-site was occupied

Electrochemical recovery of zinc from the spent pickling baths coming from the hot dip galvanizing industry. Potentiostatic operation

An electrochemical reactor was developed to recover zinc from the spent pickling solutions coming from the hot dip galvanizing industry. These solutions mainly contain ZnCl2 and FeCl2 in aqueous HCl media. The effect of the applied potential on the figures of merit (fractional conversion, current efficiency, space-time yield and specific energy consumption) of the electrochemical reactor was analysed. Voltammetric experiments were performed previously in order to select the optimum conditions to be applied in the electrolysis experiments. From the I-V curves it was inferred that bulk zinc deposition started from potential values more cathodic than -0.99 V. The hydrogen evolution reaction (HER) appeared from -0.45 V and masked the zinc cathodic peak C1, related to bulk zinc deposition, at high HCl concentrations. The presence of HCl inhibited iron deposition in synthetic samples. The additives present in the real baths, which diminish the massive hydrogen generation, allowed the observation of peak C1. The potential values to be applied in the electrolysis experiments were chosen from the voltammetric experiments and ranged between -1 V and -1.75 V. In the absence of iron in solution, as the electrode potential was shifted towards more negative values, the space-time yield of zinc and its fractional conversion increased because of the increase in the electrode roughness and the hydrogen turbulence-promoting action. Simultaneously, the specific energy consumption decreased initially due to the increase in the zinc conversion rate but decreased for the most cathodic potential value due to HER. The presence of iron in synthetic solutions led to a decrease in current efficiency associated with the reverse redox Fe 2+/Fe3+ system and to the enhancement of the HER, which also induced increments in the local pH and the subsequent zinc redissolution for the most cathodic potential values. On the contrary, the additives present in the real spent pickling baths avoided the adverse effects of iron, and zinc electrodeposition was possible even at high cathodic potential values. In fact, a potential value of -1.75 V was selected as the optimum since the conversion, the current efficiency and the space time yield obtained in the real baths were relatively high.Authors want to express their gratitude to the Universidad Politecnica de Valencia for the economical support in the project reference PAID-06-08, and to the Generalitat Valenciana for the financing of the project reference GV/2010/029.Carrillo Abad, J.; García Gabaldón, M.; Ortega Navarro, EM.; Pérez-Herranz, V. (2011). Electrochemical recovery of zinc from the spent pickling baths coming from the hot dip galvanizing industry. Potentiostatic operation. Separation and Purification Technology. 81(2):200-207. https://doi.org/10.1016/j.seppur.2011.07.029S20020781

Molecular mechanism of calcium induced trimerization of C1q-like domain of otolin-1 from human and zebrafish

Abstract The C1q superfamily includes proteins involved in innate immunity, insulin sensitivity, biomineralization and more. Among these proteins is otolin-1, which is a collagen-like protein that forms a scaffold for the biomineralization of inner ear stones in vertebrates. The globular C1q-like domain (gC1q), which is the most conserved part of otolin-1, binds Ca2+ and stabilizes its collagen-like triple helix. The molecular details of the assembly of gC1q otolin-1 trimers are not known. Here, we substituted putative Ca2+-binding acidic residues of gC1q otolin-1 with alanine to analyse how alanine influences the formation of gC1q trimers. We used human and zebrafish gC1q otolin-1 to assess how evolutionary changes affected the function of the protein. Surprisingly, the mutated forms of gC1q otolin-1 trimerized even in the absence of Ca2+, although they were less stable than native proteins saturated with Ca2+. We also found that the zebrafish gC1q domain was less stable than the human homologue under all tested conditions and became stabilized at higher concentrations of Ca2+, which showed that specific interactions leading to the neutralization of the negative charge at the axis of a gC1q trimer by Ca2+ are required for the trimers to form. Moreover, human gC1q otolin-1 seems to be optimized to function at lower concentrations of Ca2+, which is consistent with reported Ca2+ concentrations in the endolymphs of fish and mammals. Our results allow us to explain the molecular mechanism of assembly of proteins from the C1q superfamily, the modulating role of Ca2+ and expand the knowledge of biomineralization of vertebrate inner ear stones: otoliths and otoconia