Figure 1 shows a FhuA Δ1-160 based compound release system where FhuA Δ1-160 is embedded in a lipid membrane (left) together with the colorimetric HRP/TMB reporter used for quantifying TMB translocation (right).

TMB as chromogen has been developed and widely used in enzyme immunoassays (EIA) employing horseradish peroxidase [13,14]. Besides, the colorimetric HRP/TMB detection system proved to be more reproducible than the previously employed calcein assay which generates a fluorescence signal upon release of self-quenching calcein from liposomes into the surrounding solution.

The HRP/TMB detection system is based on a two step consecutive oxidative reactions A→B→C (A = TMB; B and C = first and second TMB oxidation products, see Figure 1) catalyzed by HRP in presence of hydrogen peroxide. Each single step is a pseudo-second order rate reaction with a reported second order rate constant (myeloperoxidases) [14] of: k_A→B = 3.6*10⁶ M^-1 s^-1 and k_B→C = 9.4*10⁵ M^-1 s^-1. The final TMB oxidation product C is unstable out of very acidic conditions [13] and the intermediate based on the first oxidation product B is used as reaction reporter, explaining the absorbance drop in time (see Additional file 1). The total amount of encapsulated HRP was not detectable though using the Soret absorption band. However kinetic data reproducibility was confirmed basing on a three data set for each measurement.

Figure 2 shows the six lysine residues in the FhuA Δ1-160 inner channel which upon labeling might be responsible to modulate sterically the diffusion through the channel protein. In total 12 FhuA Δ1-160 variants were generated to identify the lysine(s) which might limit TMB flux through FhuA Δ1-160 inner channel. Two subsets of six FhuA Δ1-160 variants were generated. Subset A) contains FhuA Δ1-160 variants in which one of the six lysines in the interior of FhuA Δ1-160 was substituted to alanine; subset B) contains six FhuA Δ1-160 variants in which only one lysine was not changed to alanine. Table 1 summarizes for these two subsets the TMB conversions. TMB conversions were determined by diffusion limited translocation through the FhuA Δ1-160 [12] inner channel (Additional file 1: Figure S1 and S2) using a previously reported colorimetric HRP/TMB detection system [1,13].

HRP has been entrapped in the liposome harboring FhuA Δ1-160 variants by using film hydration method coupled with extrusion. In this method, the lipid amphiphile is brought in contact with the aqueous medium containing HRP and FhuA Δ1-160 in its dry state and is subsequently hydrated to yield vesicles. After homogenization and purification of the resultant liposomes, the TMB conversion was initiated by supplementing TMB (10 μl) to the aqueous solution. Background conversions of TMB due to liposome instabilities or translocation through the membrane in absence of FhuA Δ1-160 were determined to be 13 [nM]/s (Table 1). Further control experiments were based on: liposomes harboring unlabeled FhuA Δ1-160 and the fully pyridyl-labeled FhuA Δ1-160 (starting variant). A TMB conversion of 102 [nM]/s (unlabeled FhuA Δ1-160) and 15 [nM]/s (pyridyl-labeled FhuA Δ1-160; starting variant) were reached, upon optimizing liposome preparation, and the TMB assay. The 7.9-fold higher TMB conversion in the unlabeled FhuA Δ1-160 translates an excellent detection system to monitor differences in TMB translocation through the twelve FhuA Δ1-160 variants.

The aa-position 556 has a major impact on TMB conversion: K556A substitution increases TMB conversion to 97 [nM]/s which is close to the value of the FhuA Δ1-160 unlabeled variant. A further TMB important blocking position is found by the substitution K537A increasing TMB conversion to 76 [nM]/s. In summary the following order of increased TMB conversion has been observed for subset A variants: 586 < 364 < 344 < 167 < 537 < 556.

Subset B variants of FhuA Δ1-160 have in the inner channel only one labeled pyridyl-lysine. For pyridylated position 556, a reduction of the translocation to 16 [nM]/s was achieved. The latter proves impressively that a single labeled lysine can efficiently and independently from all other labeled lysines block TMB translocation through FhuA Δ1-160. For position 537 a cooperative effect can be observed since the subset B variant shows a significantly less pronounced TMB blocking as expected from the corresponding subset A variant. Similar to the subset A) variants the following increased TMB conversion has been observed for the subset B variants: 586 > 364 > 344 > 167 > 537 > 556.

Differences in the absolute values between the two experimental data sets can likely be attributed to pyridyl labeling efficiencies, i.e. inner channel sites have a lower probability to get labeled once lysines on the protein ores are labeled or when multiple sites are labeled (Additional file 1: Figure S3). Further experimental details on the TMB conversion calculations and controls (Additional file: Figure S1 and S2), CD-spectral measurements on secondary structure stability of FhuA Δ1-160 (Additional file 1: Figure S3), size measurements of liposomes (Additional file 1: Figure S4 and S5) and simulation details, can be found in the Additional file 1.

A working hypothesis for controlling the compound flux in the inner FhuA Δ1-160 precisely is a defined and rigid conformation of the blocking lysine residue. Lysine fluctuations of all six FhuA Δ1-160 have been directly correlated to the B factors deduced from Molecular Dynamics MD trajectories in a first simulation (see Additional file 1). The B factor analysis indicates the dynamic mobility of an atom or group of atoms. The concept is derived from the X-ray scattering/crystallography theory, alternatively known as "temperature-factor" or "Debye-Waller factor" [18]. Table 1 suggests that the amino acid position 556 is a key residue in modulating the compound flux through the inner channel. Interestingly a general trend between low B factors values of the unlabeled FhuA Δ1-160 (Figure 3) and translocation importance (experimental results; Table 1), has been found. Again the most important position 556 is in the B factor analysis the least mobile one.

In detail, FhuA Δ1-160 is a β-barrel with a cross-section of 39 Å and 46 Å on the "top" part and a reduced cross-section on the "lower" exit of the barrel, 29 Å and 19 Å. K556 is placed in a rigid β-barrel at the "lower" cross-section (Figure 2) which originally interacts with the ferrichrome peptide and TonB protein [19] for further iron translocation. Other lysines are located on the opposite site K167, K537 and K586 (placed near the top cross-section) or underneath (K344; lower cross-section). As indicated by B values the positions K556, K167 and K537 have the higher blocking effects which are in accordance to the experimental results in corresponding subset A and B variants though no simulations on the pyridyl-labeled starting variant FhuA Δ1-160, has been performed (see Additional file 1 for further discussion).

In summary, experimental results and first computational simulations indicate that the rigidity of the labeled positions play an important role in generating FhuA Δ1-160 channels with a defined and "non-fluctuating" pore size. Fluctuations in pore sizes of FhuA Δ1-160 will reduce the discriminating power to control compound fluxes and are therefore an important prerequisite for a universal compound release system that can rapidly be re-engineered to match the compound size. Following up computational simulations are required to investigate in detail the roles of the pyridyl-label, to investigate cooperative effects of labeled lysine residues and taking labeling efficiency and perturbations of protein structure after labeling into account. Further FhuA Δ1-160 engineering efforts will be based on subset B) variant K556 to further advance the control of compound fluxes through the FhuA Δ1-160 channel, especially for low molecular weight compounds.

Six lysines located in the FhuA Δ1-160 channel were substituted by alanine using QuikChange (developed by Stratagene; La Jolla, CA, USA) [20] derived SDM protocol generating two subsets (A & B; Table 1) of FhuA Δ1-160 variants. Table 2 lists the primers employed for SDM.

The SDM was performed by using a two-stage PCR protocol [21]: first stage: one cycle (95°C, 1 min), three cycles (95°C, 30 s; 55°C, 1 min; 68°C, 2 min) and a second stage: one cycle (95°C, 1 min), 15 cycles (95°C, 30 s; 55°C, 1 min; 68°C, 2 min) and one cycle (68°C, 25 min). In each reaction were employed template FhuA Δ1-160 (25 ng), a primer set (see Table 2; 200 nM each), dNTP mix (200 μM) and Pfu DNA polymerase (1 U) in Pfu reaction buffer (2 × 25 μl total volume, for stage one). In stage one for each primer the extension reaction is performed in a separate PCR tube and subsequently pooled for the stage 2 PCR. In Stage 2 additional Pfu DNA polymerase (0.02 U) is supplemented before starting the second PCR. For digestion of parental DNA, DpnI (10 U; 1 h, 37°C) is supplemented to the PCR mix. All 12 FhuA Δ1-160 variants were fully sequenced to assure lysine to alanine substitutions and lack of additional mutations. Amount of DNA after PCR was quantified using a NanoDrop photometer (NanoDrop Technologies, Waltham, Massachusetts, USA).

FhuA Δ1-160 variants were expressed, extracted and purified as previously described [1] with several modifications. pPR1-FhuA Δ1-160 plasmid is freshly transformed into the expression host Escherichia coli B^E strain BL 21 (DE3) omp8 (F^- hsdS_B (r_B^- m_B^-) gal ompT dcm (DE3) Δlamb ompF::Tn5 ΔompA ΔompC) [22] . An overnight culture (TY media, 25 ml) [12] was prepared and used to inoculate expression media (inoculate 20 ml; TY medium, 250 ml) for FhuA Δ1-160 production (1-L shaking flask; 250 rpm, 37°C, 70°C humidity//Infors HT Multitron, Bottmingen, Switzerland). When the OD₅₇₈ reached 0.7, FhuA Δ1-160 protein expression was induced with IPTG (final concentration of 1 mM). Cells were grown (37°C) until the OD₅₇₈ reached 2.0-2.5 and harvested (20 min, 3220 rcf, 4°C//Eppendorf 5810R; Hamburg, Germany). Cells were resuspended in lysis buffer (12 ml; pH 8.0, 20 mM Tris, 2.5 mM MgCl₂, 0.1 mM CaCl₂, 1 mM PMSF), cooled on ice and disrupted by passing through a high-pressure homogenizer (3×, 2000 bar//Emulsiflex-C3, Avestin Inc., Ottawa, Canada). The disrupted cell suspension was mixed with FhuA Δ1-160 extraction buffer (1 ml; pH 8.0, 20 mM phosphate buffer, 2.5 mM MgCl₂, 0.1 mM CaCl₂, 20% Triton X-100) and incubated (1 h, 100 rpm, 37°C//Infors HT Multitron, Bottmingen, Switzerland). The outer membrane fractions were isolated by centrifugation (45 min, 39,700 rcf, 4°C//Avanti J-20XP, Beckman Coulter, Fullerton, USA) and resuspended in pre-solubilization buffer (9 ml; pH 8.0, 20 mM phosphate buffer, 1 mM EDTA, 0.1% oPOE, 1 mM PMSF) [23]. The resuspended outer membrane fractions were subjected to a further incubation (1 h, 200 rpm, 37°C//Infors HT Multitron, Bottmingen, Switzerland) and were subsequently isolated by centrifugation (45 min, 109,760 rcf, 4°C//Beckman Optima LE-80K Ultracentrifuge, Fullerton, USA). In the final step the isolated pellet was resuspended in solubilization buffer (9 ml; pH 8.0, 20 mM phosphate, 1 mM EDTA, 3% oPOE, 1 mM PMSF) and membrane fractions were removed by centrifugation (45 min, 109,760 rcf, 4°C//Avanti J-20XP, Beckman Coulter, Fullerton, USA). The supernatant containing FhuA Δ1-160 was concentrated using ultra-filtration (20 min, 3220 rcf, RT//Eppendorf 5810R Centricon YM30; Millipore, Bedford, USA). Purity of extracted fractions was controlled by protein gel electrophoresis and comparable to previously reported values [24]. Protein concentrations were determined using the standard BCA kit (Pierce Chemical Co, Rockford, USA).

DMSO containing 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (250 μl, 38 mM) was added drop-wise into FhuA Δ1-160 (750 μl, 4.3 μM) in phosphate buffer (pH 7.4, 0.2 M Na₂HPO₄, 0.2 M NaH₂PO₄, 3% oPOE) and stirred (1 h, 3000 rpm, RT//RCT basic IKAMAG, IKA-Werke GmbH, Staufen, Germany). Final concentration of DMSO and oPOE in the solution was 25% and 1.5%, respectively. The latter solution was used for formation of nanocompartments loaded with HRP (2.9 U/ml).

The film hydration method coupled with the mechanical dispersion technique by filter extrusion was used [25] for liposome preparation. Conventional methods of liposome production involves three basic stages: drying of the lipid solution from organic solvents, dispersion of lipids into the aqueous media, homogenization, and purification of the resultant liposomes with subsequent analysis of the final product [26]. E. coli total lipid extract (Avanti Polar Lipids, Inc., Alabaster, Alabama, USA) is a chloroform extract of the respective tissue. A mixture of E. coli total lipid extract (500 μl, 10 mg) and methanol (1:1, v/v) were used to form a thin lipid film on round-bottom flask under reduced pressure by using a rotary evaporator (Büchi Labortechnik AG, Flawil, Switzerland). The aqueous solution containing phosphate buffer (pH 7.4, 0.2 M Na₂HPO₄, 0.2 M NaH₂PO₄), FhuA Δ1-160 (3.2 μM final concentration) and HRP (2.9 U/ml) for entrapment in the interior of the vesicles was supplemented and the thin lipid film was hydrated overnight in a 30°C water bath. Nanocompartments encapsulating HRP, harboring FhuA Δ1-160 as well as amino group labeled FhuA Δ1-160 were extruded using Avanti Lipid 1 ml syringes (Alabaster, Alabama, USA), an Avanti Lipid extrusion apparatus (Alabaster, Alabama, USA) and a Bibby Heating block (Staffordshire, UK). Three polycarbonate membranes (Millipore Corporation, Bedford, MA, USA) with pore sizes of 1 μm, 0.4 μm and 0.2 μm were used with the extrusion equipment in a sequential manner to form uniform spherically shaped nanocompartments [27]. Nanocompartments were purified by gel filtration using Sepharose 4B (Fluka, Cat. no. 84962) in phosphate buffer (pH 7.4, 0.2 M Na₂HPO₄, 0.2 M NaH₂PO₄). Average diameters of nanocompartments were routinely determined using a Zeta-Sizer (Zeta-Sizer Nano Series; Malvern, Worcestershire, UK//Additional file 1: Figure S5).

TMB (Sigma Cat. N°: T 0440) assay was selected as a conversion reporter system. Pre-prepared TMB/H₂O₂ solution were used in the kinetic measurement of the TMB oxidation by the HRP [13,14]. The oxidation of TMB by the HRP/H₂O₂ system yields a blue and subsequently a yellow colored reaction product. Initial TMB oxidation kinetics are quantified by measuring an absorption maximum at 370 nm [14] using a microtiter plate reader (Omega Series; BMG LABTECH; Offenburg, Germany). TMB solution (10 μl) was supplemented to a 100 μl dispersion consisting of purified nanocompartments (in phosphate buffer, pH 7.4, 0.2 M Na₂HPO₄, 0.2 M NaH₂PO₄). Detailed kinetic results of the TMB assay of FhuA Δ1-160 variants are available in Additional file 1: Figure S1.