RESUMEN
Gravimetric transducers produce a signal based on a change in mass. These transducers can be used to construct gas sensors or biosensors using odorant binding proteins (OBPs) as recognition elements for small volatile organic compounds. The methods described in this chapter are based on the immobilization of the OBPs onto functionalized (activated) self-assembled monolayer (SAMs) on gold and on nanocrystalline diamond surfaces. Depending on the surface immobilization methods used to fabricate the biosensor, recombinant proteins can be engineered to express six histidine tags either on the N-terminal or C-terminal of the proteins and these can also be used to facilitate protein immobilization. These methods are used to produce functional sensors based on quartz crystal microbalances or surface acoustic wave devices and are also applicable to other types of gravimetric transducers.
Asunto(s)
Técnicas Biosensibles , Tecnicas de Microbalanza del Cristal de Cuarzo , Oro , Histidina , ProteínasRESUMEN
Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.
Asunto(s)
Capacidad Eléctrica , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Unión Proteica , Estereoisomerismo , Transistores ElectrónicosRESUMEN
Odorant-binding proteins (OBPs) are small soluble polypeptides found in sensory organs of vertebrates and insects as well as in secretory glands and are dedicated to detection and release of chemical stimuli. OBPs of vertebrates belong to the family of lipocalin proteins, while those of insects are folded into α-helical domains. Both types of architectures are extremely stable to temperature, organic solvents and proteolytic digestion. These characteristics make OBPs suitable elements for fabricating biosensors to be used in the environment, as well as for other biotechnological applications. The affinity of OBPs for small volatile organic compounds is in the micromolar range, and they have broad specificity to a range of ligands. For biotechnological applications, OBPs can be expressed in bacterial systems at low cost and are easily purified. The large amount of information available on their structures and affinities to different molecules should allow the design of specific mutants with desired characteristics and represent a solid base for tailoring OBPs for different applications.
Asunto(s)
Técnicas Biosensibles/métodos , Biotecnología/métodos , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Compuestos Orgánicos Volátiles/metabolismo , Secuencia de Aminoácidos , Animales , Insectos , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , VertebradosRESUMEN
OBPs have been recently demonstrated to be required for odour perception in insects and directly involved in odour discrimination. In aphids they might represent new interesting targets for the control of their population in agriculture. Based on sequence information available in the EST database, we have cloned four genes encoding odorant-binding proteins (OBP) in Acyrthosiphon pisum and homologous genes in other aphid species. Unlike OBPs from other orders of insects, that are greatly divergent, in aphids these proteins have been found to be highly conserved, with differences between species limited to only few amino acid substitutions. On the contrary, similarities between OBP sequences of the same species are poor with 31% or less of identical amino acids. Three selected OBPs (OBP1, OBP3 and OBP8) have been expressed in bacteria and purified. Ligand-binding experiments have shown similar behaviour of the three proteins towards several organic compounds, but also some significant selectivities. In particular, (E)-beta-farnesene, the alarm pheromone and its related compound farnesol exhibited good affinity to OBP3, but did not bind the other two proteins. We suggest that OBP3 could mediate response of aphids to the alarm pheromone.