Engineering of the E. coli Outer Membrane Protein FhuA to overcome the Hydrophobic Mismatch in Thick Polymeric Membranes

Background: Channel proteins like the engineered FhuA Delta 1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. Results: To increase the FhuA Delta 1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 beta-sheets, prior to the more regular periplasmatic beta-turns, were doubled leading to an extended FhuA Delta 1-159 (FhuA Delta 1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the beta-barrel folding of FhuA Delta 1-159 Ext. The FhuA.1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB1000-PEG(6000)-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). Identical experiments with the unmodified FhuA Delta 1-159 report no kinetics and presumably no insertion into the PIB1000-PEG(6000)-PIB1000 membrane. Furthermore labeling of the Lys-NH2 groups present in the FhuA Delta 1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. Conclusion: Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Delta 1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.