Sweeter and stronger: Enhancing sweetness and stability of the single chain monellin MNEI through molecular design

Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interactio

Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding.

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties

Through-bond effects in the ternary complexes of thrombin sandwiched by two DNA aptamers

Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed

Improving Protein Crystal Quality by the Without-Oil Microbatch Method: Crystallization and Preliminary X-ray Diffraction Analysis of Glutathione Synthetase from Pseudoalteromonas haloplanktis

Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from l-γ-glutamyl- l-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhGshB) has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 Å resolution and belong to space group P212121, with unit-cell parameters a = 83.28 Å, b = 119.88 Å, c = 159.82 Å. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date

Photovoltaic Pumps: Technical and Practical Aspects for Applications in Agriculture

The paper deals with a series of tests conducted on a PV-DC pump in Viterbo (42°24′ North, 12°06′ East). The tests lasted from January 2003 up to November 2004 and involved measurements of solar radiation, on both a horizontal surface and the tilted module surface, flow rates, volumes, and total dynamic heads. In total, up to 3000 data were collected every day whose analysis allowed us to find empirical relationships among system efficiencies, solar radiations, and total dynamic heads. In the second part of the paper we develop a simple method that allows both the assessment of performances of the whole system when installed in a different site from that in which the tests were performed and the optimal inclination angle of the panel to be determined in relation to annual or seasonal use (see irrigation)

Physiological and ultrastructural effects of acute ozone fumigation in the lichen Xanthoria parietina: the role of parietin and hydration state

The physiological and ultrastructural effects induced by acute exposure to ozone (O3) were investigated in the lichen Xanthoria parietina. Our working hypothesis was that parietin content and hydration of the thalli may play a role in the modulation of the effects of O3 exposure. Four batches of X. parietina samples, dry and wet, with (P+) and without (Pâ\u88\u92) parietin, were fumigated for 1 h with 3 ppm O3. The effects of O3 were assessed immediately after the fumigation and after one week of recovery under controlled conditions. O3 fumigation caused physiological and ultrastructural impairment both to the photobiont and the mycobiont, irrespective if samples were fumigated wet or dry, and P+ or Pâ\u88\u92. However, one week after fumigation, a recovery was observed in P+ samples for the photobiont and in dry samples for the mycobiont. We suggest that the hydration state may play a major role in determining the severity of the damage, while the presence of parietin may promote the recovery. Our results provide physiological and ultrastructural basis to explain the ecological insensitivity of lichens to high environmental levels of ozone occurring during dry Mediterranean summers

Manipulating Color Emission in 2D Hybrid Perovskites by Fine Tuning Halide Segregation: A Transparent Green Emitter.

Halide perovskite materials offer an ideal playground for easily tuning their color and, accordingly, the spectral range of their emitted light. In contrast to common procedures, this work demonstrates that halide substitution in Ruddlesden-Popper perovskites not only progressively modulates the bandgap, but it can also be a powerful tool to control the nanoscale phase segregation-by adjusting the halide ratio and therefore the spatial distribution of recombination centers. As a result, thin films of chloride-rich perovskite are engineered-which appear transparent to the human eye-with controlled tunable emission in the green. This is due to a rational halide substitution with iodide or bromide leading to a spatial distribution of phases where the minor component is responsible for the tunable emission, as identified by combined hyperspectral photoluminescence imaging and elemental mapping. This work paves the way for the next generation of highly tunable transparent emissive materials, which can be used as light-emitting pixels in advanced and low-cost optoelectronics