The aim of this work is the construction of a series of artificial porins with pre-defined features, to be applied in biotechnological applications. We could take advantage from the dataset of crystalized bacterial porins. These proteins form transmembrane beta barrels where each β-strand is connected to the next with alternating long loops (on the extracellular side) or short turns (on the periplasmic side). We have screened the porin sequences for conserved portions. We found a very conserved stretch encoding for two beta strands and their connecting beta turns. We thus decided to use this sequence to build multimers with rising, even number of the basic sequence (our module). The assembly of consecutive modules was first studied with bioinformatic simulations to predict the potential folding patterns. These simulations were indeed used as an additional criteria to decide the sequence of the module. Interestingly, the predicted structures formed with a number ranging from six to twelve repeats of the module showed a comparable thermodynamic stability. The molecular building of the repetitive module was based on standard molecular biology techniques. Mutagenesis of the cloned sequences was performed to polish the structure, insert tags for purification procedures and to allow the constructs to be in frame. In this way several multimers of the basic module have been obtained. They were cloned and the corresponding proteins were produced and tested in the planar bilayer membranes. Preliminary results demonstrated that the artificial pore is able to form channels in membranes. We are designing nanopores with specific diameters that can be inserted into liposomes. Their possible applications in nanotechnology for medicine will be discussed.

Generation of artificial channels by multimerization of beta-strands from natural porin

Reina S;GUARINO, FRANCESCA MARIA;DE PINTO, Vito Nicola;MESSINA, Angela Anna
2011

Abstract

The aim of this work is the construction of a series of artificial porins with pre-defined features, to be applied in biotechnological applications. We could take advantage from the dataset of crystalized bacterial porins. These proteins form transmembrane beta barrels where each β-strand is connected to the next with alternating long loops (on the extracellular side) or short turns (on the periplasmic side). We have screened the porin sequences for conserved portions. We found a very conserved stretch encoding for two beta strands and their connecting beta turns. We thus decided to use this sequence to build multimers with rising, even number of the basic sequence (our module). The assembly of consecutive modules was first studied with bioinformatic simulations to predict the potential folding patterns. These simulations were indeed used as an additional criteria to decide the sequence of the module. Interestingly, the predicted structures formed with a number ranging from six to twelve repeats of the module showed a comparable thermodynamic stability. The molecular building of the repetitive module was based on standard molecular biology techniques. Mutagenesis of the cloned sequences was performed to polish the structure, insert tags for purification procedures and to allow the constructs to be in frame. In this way several multimers of the basic module have been obtained. They were cloned and the corresponding proteins were produced and tested in the planar bilayer membranes. Preliminary results demonstrated that the artificial pore is able to form channels in membranes. We are designing nanopores with specific diameters that can be inserted into liposomes. Their possible applications in nanotechnology for medicine will be discussed.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11769/101649
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