Journal of Membrane Science & Technology

Towards preparation of chiral membranes based on barrel proteins

2^nd International Conference on Membrane Science and Technology

September 13-14, 2018 | London, UK

T Mirzaei Garakani, A Boker and U Schwaneberg

RWTH Aachen University, Germany
Fraunhofer Institute for Applied Polymer Research, Germany
Universitat Potsdam, Germany
DWI-Leibniz Institute for Interactive Materials, Germany

Scientific Tracks Abstracts: J Membr Sci Technol

Abstract:

The ability to manufacture conjugates in which polymer chains grow from the protein surface in a monodisperse fashion through controlled radical polymerization (CRP) method would bring us one step closer to generate highly functional structures. Here, we present the synthesis of so called building blocks based on transmembrane proteins (BBTP) from genetically modified ferric hydroxamate uptake protein component A (FhuA), by grafting thermoresponsive poly(N isopropylacrylamide) (PNIPAAm) from its surface. An emphasis is given on the protein engineering work to enable efficient polymerization and functionality of the designed FhuA variants. The pore-blocking cork domain was removed from naturally occurring FhuA WT by deletion of the amino acids 1-159.1,2 The naturally existing undesired lysine residues in FhuA ??CVFtev were replaced with arginine residues to maintain the charge balance.3,4 Notably, the lysines are positioned exclusively above the hydrophobic region. A water-soluble NHS-ester activated CRP initiator was chosen to be linked to the lysine residues of FhuA to form FhuA macroinitiators (MI). 2-methyl-2,4-pentanediol (MPD) was used to keep FhuA and the variants soluble in aqueous environment as well as avoid interference to the linkage of initiator units, the polymerization process and characterization techniques. FhuA variants including two recognition sites for the Tobacco Etch Virus protease were designed to generate a 6 kDa protein fragment containing one lysine residue upon the cleavage by the protease in order to facilitate analysis of the lysine modification with MALDI-ToF mass spectrometry.5 Polymerization was performed using Cu(I)Br/Me6TREN catalyst system. Analytical ultracentrifugation (AUC) indicated the presence of BBTP by a shift of the sedimentation coefficient to higher values (Figure 1A). The structural integrity of the formed FhuA -polymer conjugates was analyzed by CD spectroscopy proving that the BBTP could be synthesized without influencing the secondary structure of the membrane proteins (Figure 1B). Recent Publications: 1. Charan H, Glebe U, Anand D, Kinzel J, Zhu L, et al. (2017) Nano-thin walled micro-compartments from transmembrane protein-polymer conjugates. Soft matter 13:2866???2875. 2. Charan H, Kinzel J, Glebe U, Anand D, Mirzaei Garakani T, et al. (2016) Grafting PNIPAAm from ??-barrel shaped transmembrane nanopores. Biomaterials 107:115???123. 3. Philippart F, Arlt M, Gotzen S, Tenne J, Bocola M, et al. (2013) A hybrid ring-opening metathesis polymerization catalyst based on an engineered variant of the ?-barrel protein FhuA. Chem. Eur. J. 19: 13865???13871. 4. Güven A, Dworeck T, Fioroni M and Schwaneberg U (2011) Residue K556-A light triggerable gatekeeper to sterically control translocation in FhuA. Adv. Eng. Mater. 13:B324-B329. 5. Onaca O, Sarkar P, Roccatano D, Friedrich T, Hauer B, et al. (2008) Functionalized nanocompartments (synthosomes) with a reduction-tiggered release system. Angew. Chem. Int. Ed 47:7029???7031.

Biography :

T Mirzaei Garakani is working as project leader in group of Prof. Dr. Ulrich Schwaneberg and has her expertise in preparation and evaluation of protein-polymer hybrid membranes. Being involved in cooperative projects in highly interdisciplinary fields, made her experienced in different areas of analytical, materials and polymer chemistry, electrochemistry, bioelectrochemistry and protein biochemistry, thin film, colloid and interface chemistry as well as surface and interface characterization methods. A major part of her study is focused on ‘’Fabrication of Advanced Hybrid Functional Membranes’’ using channel Proteins. Research interest: Fabrication of hybrid membranes ● Electropolymerization ● Protein-Polymer conjugates ● Surface and interface analysis. Presenting Author's Biography: Marco Grull born on 21.08.1986,in Eberswalde – Finow in Germany. He completed his M.Sc. Biology; Major: Microbiology and Genetics, White/Red Biotechnology at RWTH Aachen University (GER). He completed his Ph.D. at the Institute of Pharmaceutical Biology (Georg-Christoph-Lichtenberg-Stipendium) at TU - Braunschweig (GER). He received his PostDoc in the Institute of Biotechnology at RWTH Aachen (GER).

E-mail: t.mirzaeigarakani@biotec.rwth-aachen.de