Molecular understanding of sterically controlled compound release through an engineered channel protein (FhuA)

Abstract

BackgroundRecently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Δ1-160; Onaca et al., 2008). Compound fluxes within the FhuA Δ1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester). Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems.ResultsIn total, 12 FhuA Δ1-160 variants were generated to gain insights on sterically controlled compound fluxes: Subset A) six FhuA Δ1-160 variants in which one of the six lysines in the interior of FhuA Δ1-160 was substituted to alanine and Subset B) six FhuA Δ1-160 variants in which only one lysine inside the barrel was not changed to alanine. Translocation efficiencies were quantified with the colorimetric TMB (3,3',5,5'-tetramethylbenzidine) detection system employing horseradish peroxidase (HRP). Investigation of the six subset A variants identified position K556A as sterically important. The K556A substitution increases TMB diffusion from 15 to 97 [nM]/s and reaches nearly the TMB diffusion value of the unlabeled FhuA Δ1-160 (102 [nM]/s). The prominent role of position K556 is confirmed by the corresponding subset B variant which contains only the K556 lysine in the interior of the barrel. Pyridyl labeling of K556 reduces TMB translocation to 16 [nM]/s reaching nearly background levels in liposomes (13 [nM]/s). A first B-factor analysis based on MD simulations confirmed that position K556 is the least fluctuating lysine among the six in the channel interior of FhuA Δ1-160 and therefore well suited for controlling compound fluxes through steric hindrance.ConclusionsA FhuA Δ1-160 based reduction triggered release system has been shown to control the compound flux by the presence of only one inner channel sterical hindrance based on 3-(2-pyridyldithio)propionic-acid labeling (amino acid position K556). As a consequence, the release kinetic can be modulated by introducing an opportune number of hindrances. The FhuA Δ1-160 channel embedded in liposomes can be advanced to a universal and compound independent release system which allows a size selective compound release through rationally re-engineered channels.

Residue K556‐A Light Triggerable Gatekeeper to Sterically Control Translocation in FhuA

“…[11] As previously found, twenty-nine lysines are present in the FhuA D1-160 with 19 lysines located on the protein surface and six inside the channel. All 29 can be labeled but only the six contained into the channel are determinant for the diffusions.…”

“…All 29 can be labeled but only the six contained into the channel are determinant for the diffusions. [11] Through computational B-factor analysis it was found that amino acid position K556 is the least fluctuating lysine, well positioned in the smaller channel ore and most effective in flux control upon labeling. For designing a light triggered system with a precise steric control we decided to test the K556-hypothesis by generating a FhuA D1-160 variant with only one lysine in the barrel interior at position 556 and to label the lysine with NVOC-Cl.…”

“…[34] In a further study six lysine which are located inside the FhuA channel protein were extensively mutated (12 variants) to identify position 556 as being most important for controlling compound flux through the FhuA channel protein by steric hindrance. [11] Computational B-factor analysis confirmed that K556 is the least fluctuating lysine among the six in the FhuA barrel interior. [11] FhuA is a large monomeric, two-domain transmembrane protein (79 kDa) located in the Escherichia coli outer membrane, folded into 22 anti-parallel b-strands.…”

“…[11] Computational B-factor analysis confirmed that K556 is the least fluctuating lysine among the six in the FhuA barrel interior. [11] FhuA is a large monomeric, two-domain transmembrane protein (79 kDa) located in the Escherichia coli outer membrane, folded into 22 anti-parallel b-strands. [35] By removing the ''cork'' domain (deletion of amino acids 5-160 [36] ) a deletion variant had been generated which behaves like a large passive diffusion channel (FhuA D1-160) [37] allowing even the translocation of single stranded DNA.…”

“…The conversion of the HRP substrate TMB [11] is determined using well established and repeatedly reported protocols. [11,37,42] HRP oxidizes TMB in two subsequent steps to the corresponding diimine (A ! B !…”