Electronic Olfactory Sensor Based on A.†mellifera Odorant-Binding Protein†14 on a Reduced Graphene Oxide Field-Effect Transistor.

An olfactory biosensor based on a reduced graphene oxide (rGO) field-effect transistor (FET), functionalized by the odorant-binding protein 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the in situ and real-time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand-receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K(d)=4 µM to K(d)=3.3 mM. The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.

Insights into structural features determining odorant affinities to honey bee odorant binding protein 14.

Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant.

Honey bee odorant-binding protein 14: effects on thermal stability upon odorant binding revealed by FT-IR spectroscopy and CD measurements.

In the present work, we study the effect of odorant binding on the thermal stability of honey bee (Apis mellifera L.) odorant-binding protein 14. Thermal denaturation of the protein in the absence and presence of different odorant molecules was monitored by Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). FT-IR spectra show characteristic bands for intermolecular aggregation through the formation of intermolecular ß-sheets during the heating process. Transition temperatures in the FT-IR spectra were evaluated using moving-window 2D correlation maps and confirmed by CD measurements. The obtained results reveal an increase of the denaturation temperature of the protein when bound to an odorant molecule. We could also discriminate between high- and low-affinity odorants by determining transition temperatures, as demonstrated independently by the two applied methodologies. The increased thermal stability in the presence of ligands is attributed to a stabilizing effect of non-covalent interactions between odorant-binding protein 14 and the odorant molecule.

Recombinant production of an Aspergillus nidulans class I hydrophobin (DewA) in Hypocrea jecorina (Trichoderma reesei) is promoter-dependent.

Fungal hydrophobins have potential for several applications because of their abilities to change the hydrophobicity of different surfaces. Yet because of their tendency for aggregation and attachment to interfacial areas only few production processes have so far been reported. Towards the development of a heterologous production system, we report here the expression of a class I hydrophobin DewA of Aspergillus nidulans in Hypocrea jecorina (Trichoderma reesei). Using the H. jecorina hfb2 (class II hydrophobin-encoding) promoter and lactose as a carbon source, only a minor fraction of the DewA remained cell-wall-bound and the majority of it secreted into the medium with up to 15% of the total secreted protein. N-terminal amino acid sequencing showed that it was correctly processed. In contrast, no DewA was produced under the cel7A (cellobiohydrolase I) promoter, although its mRNA was abundantly detected in the cells. This lack of secretion is not due to trapping in the cell wall or to its degradation because of the unfolded protein response. Recombinant DewA could be conveniently precipitated from the culture filtrate, and its bioactivity proven by its ability to stably bind to hydrophilic and hydrophobic surfaces (glass and Teflon, respectively). We thus consider H. jecorina as a promising host for further optimization of DewA production.

Electronic Biosensing with Functionalized rGO FETs.

In the following we give a short summary of examples for biosensor concepts in areas in which reduced graphene oxide-based electronic devices can be developed into new classes of biosensors, which are highly sensitive, label-free, disposable and cheap, with electronic signals that are easy to analyze and interpret, suitable for multiplexed operation and for remote control, compatible with NFC technology, etc., and in many cases a clear and promising alternative to optical sensors. The presented areas concern sensing challenges in medical diagnostics with an example for detecting general antibody-antigen interactions, for the monitoring of toxins and pathogens in food and feed stuff, exemplified by the detection of aflatoxins, and the area of smell sensors, which are certainly the most exciting development as there are very few existing examples in which the typically small and hydrophobic odorant molecules can be detected by other means. The example given here concerns the recording of a honey flavor (and a cancer marker for neuroblastoma), homovanillic acid, by the odorant binding protein OBP 14 from the honey bee, immobilized on the reduced graphene oxide gate of an FET sensor.

Dietary magnesium restriction reduces amygdala-hypothalamic GluN1 receptor complex levels in mice.

Reduced daily intake of magnesium (Mg(2+)) is suggested to contribute to depression. Indeed, preclinical studies show dietary magnesium restriction (MgR) elicits enhanced depression-like behaviour establishing a causal relationship. Amongst other mechanisms, Mg(2+) gates the activity of N-methyl-D-asparte (NMDA) receptors; however, it is not known whether reduced dietary Mg(2+) intake can indeed affect brain NMDA receptor complexes. Thus, the aim of the current study was to reveal whether MgR induces changes in brain NMDA receptor subunit composition that would indicate altered NMDA receptor regulation. The results revealed that enhanced depression-like behaviour elicited by MgR was associated with reduced amygdala-hypothalamic protein levels of GluN1-containing NMDA complexes. No change in GluN1 mRNA levels was observed indicating posttranslational changes were induced by dietary Mg(2+) restriction. To reveal possible protein interaction partners, GluN1 immunoprecipitation and proximity ligation assays were carried out revealing the expected GluN1 subunit association with GluN2A, GluN2B, but also novel interactions with GluA1, GluA2 in addition to known downstream signalling proteins. Chronic paroxetine treatment in MgR mice normalized enhanced depression-like behaviour, but did not alter protein levels of GluN1-containing NMDA receptors, indicating targets downstream of the NMDA receptor. Collectively, present data demonstrate that dietary MgR alters brain levels of GluN1-containing NMDA receptor complexes, containing GluN2A, GluN2B, AMPA receptors GluA1, GluA2 and several protein kinases. These data indicate that the modulation of dietary Mg(2+) intake may alter the function and signalling of this receptor complex indicating its involvement in the enhanced depression-like behaviour elicited by MgR.