Justesenhjort9100

The development of an "electronic tongue" based on an EGOFET device coupled to odorant binding proteins (OBPs) for enantiomers differentiation is presented.Olfaction is capable of accomplishing incredible tasks it starts with capturing an odor molecule, delivering it to the odorant receptors, converting it into an electrical stimulus and transmitting the data to the brain. And all of this in milliseconds. The sense of smell is not yet fully decoded and is far from being replicated by modern sensor technologies. One approach to convert biological recognition- and binding events in real-time and in a label-free manner to electrical signals is emulated in a "biomimetic electronic smell sensor". It is based on a transistor, in many cases realized as a field-effect transistor (FET) with a biorecognition element, e.g., an odorant binding protein (OBP) converting the binding event of one of its typically many ligands directly into a measurable electrical signal. OBPs are immobilized on these FETs and modulate the current in the presence of smell molecules due to the charge redistribution in the gated channel. Graphene is an elegant candidate to realize such a sensor device because an atomic monolayer of a semiconducting material leads to increased sensitivity. Beside the direct molecule interaction with the substrate upon binding and its excellent biocompatible character, graphene has the advantage of a biological-friendly working point in the sub-Volt regime. Different approaches of preparation and functionalization of graphene field-effect transistors (gFETs) are utilized to tune the performance for odorant sensing. The evaluation of kinetic binding parameters like association and dissociation rate constants and the equilibrium affinity constants of protein-ligand interactions can be derived from the direct electrical read-out of such miniaturized sensor systems. In this article, the state of the art of gFET preparation, functionalization, and operation for odorant sensing will be discussed.Understanding physiological tasks may start by a good understanding of evolutionary traits in a given protein gene family. The successful completion of various genome projects is a key step forward for comparative analysis of similar and/or orthologous genes between species, measuring genetic relatednesses, studying evolutionary changes among different behaviors, helping to identify specifically conserved genes or genes that are variable enough to determine a new strain or organism. How evolutionary data can improve the understanding of a protein gene family is exploited here in the case of the insect Chemosensory Proteins (CSPs) as a guidance model. In this chapter, Diptera are presented not only as a huge diversity of appearances (phenotypes), behaviors and lifestyles, but also as major differences that are gene copy numbers, gene family members, polymorphisms and evolutionary rate variations, without any a priori assumption about the origin and function of the sampled gene family.The development of sensors that mimic the natural smell sensing mechanism and selectively recognizes the odorants remains highly challenging. #link# Electrochemical based sensing approaches aiming at monitoring molecular recognition events between surface receptors and analytes in solution or in the gas phase, are one possible transduction platforms among others for the construction of an artificial nose. The principle of electrochemical detection lies on the shift of the potential/current during the recognition event, which is proportional to the concentration of the analyte, in our case the odorant. A tremendous amount of efforts has been put into making electrochemical sensors sensitive and selective to the analyte of interest through the use of nanomaterials, development of different detection schemes and application of innovative receptor ligands for selective detection of the analyte. There have been significant advances in electrochemical based odorant sensing by using odorant binding proteins (OBP) as surface receptors, small soluble proteins present in nasal mucus at millimolar concentrations where the hydrophobic binding pocket gives the ability to reversibly bind odorant molecules. As OBPs are robust and easy to produce receptors, they are good candidates for the design of biosensors. In this chapter, we focus on the progress made on the detection of odorant molecules using OBPs as a bioreceptor and electrochemistry as a transduction method.Pheromone binding proteins (PBPs) are small soluble proteins (about 15kDa) that play striking roles in the detection of sex pheromones in insects. Many studies including structural analysis, binding simulation, and in vitro assays have been performed to clarify the modes of action of PBPs. Although these studies have provided valuable contributions toward the understanding of which key amino acid components contribute to the correct folding of PBPs and their binding affinities to sex pheromones, the functional characteristics of PBPs in the natural environment is still obscure. Recent developments in genome editing have begun to enable the functional examination of PBPs in in vivo. Among insect PBPs, BmPBP1 is one of the most well-characterized, there being rich understanding of its structure, biochemical analysis, binding affinity, localization, and the relationship between the type of olfactory receptors and its expression. A recent study has shown that BmPBP1 contributes sensitivity, but not selectivity of sex pheromone detection in the silkmoth Bombyx mori. In this chapter, based on a current report of the functional characterization of BmPBP1 using genome editing, we provide one example of a useful analytical method to clarify the functional role of PBP in vivo.Modifying the affinity of odorant-binding proteins (OBPs) to small ligands by replacement of specific residues in the binding pocket may lead to several technological applications. CP-91149 to their compact and stable structures, OBPs are currently regarded as the best candidates to be used in biosensing elements for odorants and volatiles detection. The wide and rich information on the structure of these proteins both in their apo-forms and in complexes with specific ligands provides guidelines to design reliable mutants to monitor specific targets. The same engineered proteins may also find applications in the slow release of pheromones and other chemicals in the environment, as well as in the fine purification of drugs, including the resolution of racemates. Apart from such useful applications, site-directed mutagenesis represents an interesting approach to dissect the specific interactions between small chemicals and amino acid residues in the binding pocket. These studies can lead to design of better ligands, such as pheromone analogues with desired physico-chemical characteristics. In this chapter we examine the different uses of mutagenesis applied to OBPs and report a couple of protocols that have been successful in our hands.The technique of two-electrode voltage-clamp (TEVC) recording from the heterologous expression system of olfactory receptors (ORs) in Xenopus laevis oocytes has been widely used to deorphanize insect ORs, that is to identify specific ligands for each of them. However, there is a controversial issue on whether ORs are activated by the odorant/OBP complex or the odorant alone. The mechanism of interaction among odorants, odorant-binding proteins (OBPs) and ORs remains largely unknown, due to the limitations in the use of scientific and innovative methods. In this chapter, the modified Xenopus oocytes expression system combined with TEVC technique is used to approach this issue. We describe the experimental strategies and provide detailed protocols for recording the signals generated by ORs in response to odorant/OBP complex at different concentrations. Results obtained by this approach have revealed that the presence of OBPs in the system affects the selectivity and sensitivity responses of ORs. Such studies help understanding the molecular mechanism of odorant detection in peripheral nervous system.Fine structure immunocytochemistry enables the in situ localization of odorant-binding proteins with the high resolution of the electron microscope. Protocols for successfully labeling these proteins in insect chemosensory sensilla are given and some pitfalls pinpointed as well as ways to avoid them. The major achievements accomplished by these methods are briefly reviewed.Assessing the ligand-binding properties of OBPs and CSPs is essential for understanding their physiological function. It also provides basic information when these proteins are used as biosensing elements for instrumental measurement of odors. Although different approaches have been applied in the past to evaluate the affinity of receptors and soluble binding proteins to their ligands, using a fluorescent reporter represents the method of choice for OBPs and CSPs. link2 It offers the advantages of working at the equilibrium, being simple, fast and inexpensive, without requiring the use of radioactive tracers. link3 However, as an indirect method, the fluorescence competitive binding approach presents drawbacks and sometimes requires an elaborate analysis to explain unexpected results. Here, after a brief survey of the different approaches to evaluate affinity constants, we focus on the fluorescence binding assay as applied to OBPs and CSPs, discussing situations that may require closer inspection of the results.Obtaining information about functional details of proteins of extinct species is of critical importance for a better understanding of the real-life appearance, behavior and ecology of these lost entries in the book of life. In this chapter, we discuss the possibilities to retrieve the necessary DNA sequence information from paleogenomic data obtained from fossil specimens, which can then be used to express and subsequently analyze the protein of interest. We discuss the problems specific to ancient DNA, including miscoding lesions, short read length and incomplete paleogenome assemblies. Finally, we discuss an alternative, but currently rarely used approach, direct PCR amplification, which is especially useful for comparatively short proteins.Odorant binding proteins (OBPs) are small proteins, some of which bind odorants with high specificity. OBPs are relatively easy to produce and show a pronounced stability toward thermal and chemical denaturation. This high stability renders OBPs attractive candidates for the development of odorant detections systems. Unfortunately, binding of odorants is not easy to quantify due to lack of spectroscopic signals upon binding. Therefore, a possible approach to detect binding is to employ the shift in thermal or chemical stability upon ligand-protein interaction. Being a rather indirect approach, the experimental setup should be done with care. Here, the experimental results on stability of OBPs are summarized and issues which should be considered when performing stability experiments are discussed.Insect odorant binding proteins (OBPs) and chemosensory proteins (CSPs) are proteins deputed to the solubilization, transport and stabilization of lipophilic and odorant compounds. These proteins have a conserved fold, which undergoes massive structural rearrangements in order to accommodate medium to large-sized lipophilic ligands. Solution NMR spectroscopy, due to its intrinsically dynamic nature, is the perfect technique to extrapolate structural information and dynamic parameters and to elucidate the conformational changes that occur upon ligand binding. This chapter will describe in detail the experimental protocols for the production and purification of isotope-labeled recombinant CSPs and OBPs for NMR studies. Detailed procedures for spectra acquisition, processing and analysis will be presented, focusing on the protein CSP-sg4 from Schistocerca gregaria as a model. Finally, experiments aimed at providing information on protein flexibility and ligand binding modes will also be described.