@article{CharanKinzelGlebeetal.2016, author = {Charan, Himanshu and Kinzel, Julia and Glebe, Ulrich and Anand, Deepak and Garakani, Tayebeh Mirzaei and Zhu, Leilei and Bocola, Marco and Schwaneberg, Ulrich and B{\"o}ker, Alexander}, title = {Grafting PNIPAAm from beta-barrel shaped transmembrane nanopores}, series = {Biomaterials : biomaterials reviews online}, volume = {107}, journal = {Biomaterials : biomaterials reviews online}, publisher = {Elsevier}, address = {Oxford}, issn = {0142-9612}, doi = {10.1016/j.biomaterials.2016.08.033}, pages = {115 -- 123}, year = {2016}, abstract = {The research on protein-polymer conjugates by grafting from the surface of proteins has gained significant interest in the last decade. While there are many studies with globular proteins, membrane proteins have remained untouched to the best of our knowledge. In this study, we established the conjugate formation with a class of transmembrane proteins and grow polymer chains from the ferric hydroxamate uptake protein component A (FhuA; a beta-barrel transmembrane protein of Escherichia coli). As the lysine residues of naturally occurring FhuA are distributed over the whole protein, FhuA was reengineered to have up to 11 lysines, distributed symmetrically in a rim on the membrane exposed side (outside) of the protein channel and exclusively above the hydrophobic region. Reengineering of FhuA ensures a polymer growth only on the outside of the beta-barrel and prevents blockage of the channel as a result of the polymerization. A water-soluble initiator for controlled radical polymerization (CRP) was consecutively linked to the lysine residues of FhuA and N-isopropylacrylamide (NIPAAm) polymerized under copper mediated CRP conditions. The conjugate formation was analyzed by using MALDI-ToF mass spectrometry, SDS-PAGE, circular dichroism spectroscopy, analytical ultracentrifugation, dynamic light scattering, transmission electron microscopy and size exclusion chromatography. Such conjugates combine the specific functions of the transmembrane proteins, like maintaining membrane potential gradients or translocation of substrates with the unique properties of synthetic polymers such as temperature and pH stimuli handles. FhuA-PNIPAAm conjugates will serve as functional nanosized building blocks for applications in targeted drug delivery, self-assembly systems, functional membranes and transmembrane protein gated nanoreactors. (C) 2016 Elsevier Ltd. All rights reserved.}, language = {en} } @article{GarakaniLiuGlebeetal.2019, author = {Garakani, Tayebeh Mirzaei and Liu, Zhanzhi and Glebe, Ulrich and Gehrmann, Julia and Lazar, Jaroslav and Mertens, Marie Anna Stephanie and M{\"o}ller, Mieke and Hamzelui, Niloofar and Zhu, Leilei and Schnakenberg, Uwe and B{\"o}ker, Alexander and Schwaneberg, Ulrich}, title = {In Situ Monitoring of Membrane Protein Insertion into Block Copolymer Vesicle Membranes and Their Spreading via Potential-Assisted Approach}, series = {ACS applied materials \& interfaces}, volume = {11}, journal = {ACS applied materials \& interfaces}, number = {32}, publisher = {American Chemical Society}, address = {Washington}, issn = {1944-8244}, doi = {10.1021/acsami.9b09302}, pages = {29276 -- 29289}, year = {2019}, abstract = {Synthosomes are polymer vesicles with trans membrane proteins incorporated into block copolymer membranes. They have been used for selective transport in or out of the vesicles as well as catalysis inside the compartments. However, both the insertion process of the membrane protein, forming nanopores, and the spreading of the vesicles on planar substrates to form solid-supported biomimetic membranes have been rarely studied yet. Herein, we address these two points and, first, shed light on the real-time monitoring of protein insertion via isothermal titration calorimetry. Second, the spreading process on different solid supports, namely, SiO2, glass, and gold, via different techniques like spin- and dip-coating as well as a completely new approach of potential-assisted spreading on gold surfaces was studied. While inhomogeneous layers occur via traditional methods, our proposed potential-assisted strategy to induce adsorption of positively charged vesicles by applying negative potential on the electrode leads to remarkable vesicle spreading and their further fusion to form more homogeneous planar copolymer films on gold. The polymer vesicles in our study are formed from amphiphilic copolymers poly(2-methyl oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl oxazoline) (PMOXA-b-PDMS-b-PMOXA). Engineered variants of the transmembrane protein ferric hydroxamate uptake protein component A (FhuA), one of the largest beta-barrel channel proteins, are used as model nanopores. The incorporation of FhuA Delta 1-160 is shown to facilitate the vesicle spreading process further. Moreover, high accessibility of cysteine inside the channel was proven by linkage of a fluorescent dye inside the engineered variant FhuA Delta CVFtev and hence preserved functionality of the channels after spreading. The porosity and functionality of the spread synthosomes on the gold plates have been examined by studying the passive ion transport response in the presence of Li+ and ClO4- ions and electrochemical impedance spectroscopy analysis. Our approach to form solid-supported biomimetic membranes via the potential-assisted strategy could be important for the development of new (bio-) sensors and membranes.}, language = {en} } @article{CharanGlebeAnandetal.2017, author = {Charan, Himanshu and Glebe, Ulrich and Anand, Deepak and Kinzel, Julia and Zhu, Leilei and Bocola, Marco and Garakani, Tayebeh Mirzaei and Schwaneberg, Ulrich and B{\"o}ker, Alexander}, title = {Nano-thin walled micro-compartments from transmembrane protein-polymer conjugates}, series = {Soft matter}, volume = {13}, journal = {Soft matter}, publisher = {Royal Society of Chemistry}, address = {Cambridge}, issn = {1744-683X}, doi = {10.1039/c6sm02520j}, pages = {2866 -- 2875}, year = {2017}, abstract = {The high interfacial activity of protein-polymer conjugates has inspired their use as stabilizers for Pickering emulsions, resulting in many interesting applications such as synthesis of templated micro-compartments and protocells or vehicles for drug and gene delivery. In this study we report, for the first time, the stabilization of Pickering emulsions with conjugates of a genetically modified transmembrane protein, ferric hydroxamate uptake protein component A (FhuA). The lysine residues of FhuA with open pore (FhuA Delta CVFtev) were modified to attach an initiator and consequently controlled radical polymerization (CRP) carried out via the grafting-from technique. The resulting conjugates of FhuA Delta CVFtev with poly(N-isopropylacrylamide) (PNIPAAm) and poly((2-dimethylamino) ethyl methacrylate) (PDMAEMA), the so-called building blocks based on transmembrane proteins (BBTP), have been shown to engender larger structures. The properties such as pH-responsivity, temperature-responsivity and interfacial activity of the BBTP were analyzed using UV-Vis spectrophotometry and pendant drop tensiometry. The BBTP were then utilized for the synthesis of highly stable Pickering emulsions, which could remain non-coalesced for well over a month. A new UV-crosslinkable monomer was synthesized and copolymerized with NIPAAm from the protein. The emulsion droplets, upon crosslinking of polymer chains, yielded micro-compartments. Fluorescence microscopy proved that these compartments are of micrometer scale, while cryo-scanning electron microscopy and scanning force microscopy analysis yielded a thickness in the range of 11.1 +/- 0.6 to 38.0 +/- 18.2 nm for the stabilizing layer of the conjugates. Such micro-compartments would prove to be beneficial in drug delivery applications, owing to the possibility of using the channel of the transmembrane protein as a gate and the smart polymer chains as trigger switches to tune the behavior of the capsules.}, language = {en} }