Insertion of Cecropin A and reconstitution of bacterial outer membrane protein FhuA variants in polymeric membranes
Year: 2011
Type: dissertation
Abstract: Polymer based nanocompartments (polymersomes) have potential applications in synthetic biology (pathway engineering), medicine (drug release), and industrial biotechnology (chiral nanoreactors, multistep synthesis, bioconversions in non-aqueous environments, and selective product recovery). The aforementioned goals can be accomplished by polymer membrane functionalization through covalent bonding or inclusion of proteins/peptides, to obtain specific properties like recognition, catalytic activity and facilitated diffusion, mimicking the complexity of a biological membrane. Membrane protein FhuA Delta1-159 is already reported to be functionally reconstituted as channel protein into polymersome membranes of some polymers like PMOXA-PDMS-PMOXA. Nevertheless the hydrophobic mismatch, defined as the difference between the hydrophobic length of a membrane protein and the hydrophobic thickness of the membrane it spans, prevents the insertion of channel proteins like the engineered FhuA Delta1-159 into thick polymeric membranes. The first part of this thesis deals with addressing the challenge to minimize the hydrophobic mismatch between the channel protein and the polymeric membrane. A “rational” strategy to double the last five amino acids of each of the 22 beta-sheets present in FhuA Delta1-159 prior to the more regular periplasmatic beta-turns has been developed (FhuA Delta1-159 Ext). As a result the length of the calculated protein’s hydrophobic portion was increased by 1 nm. The secondary structure prediction and Circular Dichroism (CD) spectroscopy strongly suggest the beta-barrel structure of the engineered protein. The protein insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB1000-PEG6000-PIB1000 (PIB = polyisobutylene, PEG = polyethyleneglycol) has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine). Similar experiments with the parent FhuA Delta1-159 protein showed no insertion into the PIB1000-PEG6000-PIB1000 membrane. Furthermore labeling of the Lys-NH2 groups present in the FhuA Delta1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. The FhuA Delta1-159 Ext flexibility opens further possibilities to be applied as nano-channel in different block copolymer vesicles. In the thesis’s second part, a fusion protein (Cecropin A-EGFP) based on the Cecropin A antibacterial peptide and the EGFP (Enhanced Green Fluorescent Protein) was designed, expressed and biophysically characterized for the functionalization of a polymersome surface. The interaction with the tri-block PIB-PEG-PIB based polymersome membrane was analyzed by CD, coupled with EGFP and Trp fluorescence measurements. Results revealed that the Cecropin A peptide is inserted into the polymersome membrane mimicking its lipid bilayer behavior and acting as “membrane surface anchor” for water soluble proteins.
Source: RWTH Publications (RWTH Aachen)
Authors: Noor Muhammad, Ulrich Schwaneberg
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2216633729