Grafting PNIPAAm from β-barrel shaped transmembrane nanopores.
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 β-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 β-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.
Fraunhofer Institute for Applied Polymer Research IAP, Geiselbergstr. 69, 14476, Potsdam-Golm, Germany; Lehrstuhl für Polymermaterialien und Polymertechnologie, Universität Potsdam, 14476, Potsdam-Golm, Germany.
Himanshu
H
Charan
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074, Aachen, Germany.
Julia
J
Kinzel
Fraunhofer Institute for Applied Polymer Research IAP, Geiselbergstr. 69, 14476, Potsdam-Golm, Germany.
Ulrich
U
Glebe
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074, Aachen, Germany.
Deepak
D
Anand
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074, Aachen, Germany; DWI - Leibniz Institute for Interactive Materials e.V., Forckenbeckstr. 50, 52056, Aachen, Germany.
Tayebeh Mirzaei
TM
Garakani
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074, Aachen, Germany.
Leilei
L
Zhu
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074, Aachen, Germany.
Marco
M
Bocola
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074, Aachen, Germany; DWI - Leibniz Institute for Interactive Materials e.V., Forckenbeckstr. 50, 52056, Aachen, Germany. Electronic address: u.schwaneberg@biotec.rwth-aachen.de.
Ulrich
U
Schwaneberg
Fraunhofer Institute for Applied Polymer Research IAP, Geiselbergstr. 69, 14476, Potsdam-Golm, Germany; Lehrstuhl für Polymermaterialien und Polymertechnologie, Universität Potsdam, 14476, Potsdam-Golm, Germany. Electronic address: alexander.boeker@iap.fraunhofer.de.
Alexander
A
Böker
D000180:Acrylic Resins / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D008051:Lipid Bilayers / Q000737:chemistry*; D008968:Molecular Conformation; D058608:Nanopores / Q000648:ultrastructure*; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship
D000180:Acrylic Resins; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008051:Lipid Bilayers; C052970:poly-N-isopropylacrylamide
BBTP; FhuA; Grafting-from polymerization; NIPAAm; Protein-polymer conjugate; Transmembrane protein