Project

polymer nanovesicles; switchable transport; engineered channel proteins; block copolymers

Lead
Lay summary
The aim of this project is to design and engineer channel proteins that can act as controllable gates once embedded in the membrane of nanovesicles. The reversible control of the transport of molecules through the channel protein can be established by changes in pH, temperature or light. OmpF channel protein has been chosen as model channel protein due to its thermal and chemical stability. The OmpF channel protein will be equipped with polymers that are pH or temperature sensitive. The reversibility of the switch introduced inside the channel proteins' pores will be characterized once the channel proteins are embedded in the wall of polymer nanovesicles. The polymeric nanovesicles are created by self-assembly of amphiphilic block copolymers in water. Amphiphilic block copolymers are made of a hydrophobic block and a hydrophilic block and they spontaneously form vesicles in water. The polymeric systems are a novel alternative to the liposome carriers, and are more stable, while preserving all the advantages of lipidic systems, such as lack of immunogenicity. The obtained vesicles reconstituting engineered channel proteins will be characterized using fluorescence and spectroscopic methods. The pH/temperature-sensitive OmpF hybrid system will be a first step in mimicking nature (e.g. ligand gated channels like acetylcholine receptors, light gated like channel rhodopsin) to produce functional proteins with potential applications in biotechnology or biomedicine. We anticipate that channel proteins engineered for reversible transport induced by changes in external stimuli (pH, temperature, light) will be used for tunable release of the content of liposomes or polymeric vesicles. This general system will be of special interest for various biomedical, biotechnological and biosensing applications. A biomedical application would be polymeric vesicles containing drugs or drug producing enzymes inside and embedded channel proteins that allow switchable transport of the prodrug/drug across the polymer membrane. With this specific system a precise control of the rate and the moment of the release of the drugs could be achieved.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

In biological membranes channel proteins facilitate the transport of different molecules into and out of the cell. The transport of substances can be active transport or passive diffusion. Channel proteins like OmpF and Opdk which are found in the outer membrane of Escherichia coli and Pseudomonas aeruginosa, respectively, act as passive gateways for different small molecules (less than 600 Da). However in biological cell membranes they do not undergo conformational changes for increased selectivity in molecule transport.The aim of the project is to engineer channel proteins in order to obtain a reversible switchable transport across them. To achieve this, we plan to modify such unspecific channel proteins by introducing cysteines into the genes of the channels proteins. Responsive polymer chains that can respond to external stimuli such as temperature, light, pH, presence of ions, will be chemically attached to or grown from the introduced cysteines in order to constrict the channel. Upon appropriate exterior input, the polymer chain will change its solubility resulting in blockage of the channel pore, while restoring the initial conditions will cause the polymer to assume its earlier form of solubility and thus reopening the channel protein. By modulation of the polymer chain length it is possible to control the cutoff of the pore, transforming it in a molecular sieve for substances in certain molecular weight ranges. To the best of our knowledge, the approach for triggered transport across channel proteins would be the first of its kind for biological pores and will therefore offer a general strategy for obtaining reversible biological pores. Moreover we envisage that a blockage of the channel proteins with sensitive peptides (pH, temperature) introduced using genetic engineering methods will offer an alternative to the use of synthetic polymers for blocking.The reversibility of the switch introduced inside the channel proteins` pores will be characterized once the channel proteins are reconstituted in polymer nanovesicles. The polymeric nanovesicles are created by self assembly of amphiphilic block copolymers, for e.g poly-(2-methyloxazoline)-poly (dimethylsiloxane)-poly (2-methyloxazoline). The polymeric systems are a novel alternative to the liposome carriers, and are more stable, while preserving all the advantages of lipidic systems, such as lack of immunogenicity. The polymeric vesicles reconstituting engineered channel proteins will be characterized using fluorescence and spectroscopic methods. We anticipate that channel proteins engineered for reversible, controlled transport caused by changes in external stimulation (pH, temperature, light) will be used in drug delivery for tunable release of the content of liposomes or polymeric vesicles, as well as in biotechnological applications or biosensing.