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BackgroundChannel proteins like the engineered FhuA Δ1-159 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.ResultsTo increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext 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). Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB1000-PEG6000-PIB1000 membrane. Furthermore labeling of the Lys-NH2 groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer.ConclusionUsing 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 Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

BackgroundChannel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering.ResultsThe FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%.ConclusionIn this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

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The general aim of our work is to build a set of engineered protein channels to be used in liposome and polymersome technology with a special emphasis in delivery applications. The channel proteins FhuA and OmpF are modified to answer to chemical (Angew. Chem. Int. Ed., 2008), pH (Soft Matter, 2011), and light stimuli. In this study a first light triggered release system is developed by employing the photo‐cleavable label 6‐nitroveratryloxycarbonyl chloride (NVOC‐Cl) and FhuA variants with six, five, and only one lysine in the barrel. Kinetic studies on liposome inserted FhuA variants, using 3,3′,5,5′‐tetramethylbenzidine (TMB)/horseradish peroxidase (HRP) as detection system led to the discovery of a single labeled amino acid position, K556, that is sufficient to act as a gate and that controls TMB translocation through the FhuA Δ1‐160 pore. Background conversion of TMB in the absence of FhuA Δ1‐160 ranges from 13 (non‐photo‐irradiated) to 27 (photo‐irradiated) n · s−1. A “fully” open FhuA Δ1‐160 channel reaches TMB conversions up to 113 × 10−9 M · s−1; a “fully” labeled FhuA Δ1‐160 shows a TMB conversion of 29 × 10−9 M · s−1 which is close to background levels. The engineered FhuA Δ1‐160 with only one lysine in the barrel interior (K556) shows a TMB conversion of 33 × 10−9 M · s−1 after labeling and after NVOC photo‐cleavage a conversion of 94 × 10−9 M · s−1. The latter proves the gate keeping role of position 556 in sterically modulating TMB fluxes. CD spectra, cryogenic TEM, and DLS experiments were performed to characterize the employed liposomes with embedded FhuA Δ1‐160 variants.