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

Güven A, Fioroni M, Hauer B, Schwaneberg U - J Nanobiotechnology (2010)

Bottom Line: Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Delta1-160; Onaca et al., 2008).Pyridyl labeling of K556 reduces TMB translocation to 16 [nM]/s reaching nearly background levels in liposomes (13 [nM]/s).A FhuA Delta1-160 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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. u.schwaneberg@biotec.rwth-aachen.de.

ABSTRACT

Background: Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Delta1-160; Onaca et al., 2008). Compound fluxes within the FhuA Delta1-160 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.

Results: In total, 12 FhuA Delta1-160 variants were generated to gain insights on sterically controlled compound fluxes: Subset A) six FhuA Delta1-160 variants in which one of the six lysines in the interior of FhuA Delta1-160 was substituted to alanine and Subset B) six FhuA Delta1-160 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 Delta1-160 (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 Delta1-160 and therefore well suited for controlling compound fluxes through steric hindrance.

Conclusions: A FhuA Delta1-160 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 Delta1-160 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.

No MeSH data available.


Schematic representation of functionalized liposome system. The FhuA Δ1-160 channel protein embedded in the liposomal lipid membrane (left panel) employed as reduction triggered gateway for the in/out diffusion of TMB and hydrogen peroxide (right panel) used in the HRP/TMB colorimetric assay.
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Figure 1: Schematic representation of functionalized liposome system. The FhuA Δ1-160 channel protein embedded in the liposomal lipid membrane (left panel) employed as reduction triggered gateway for the in/out diffusion of TMB and hydrogen peroxide (right panel) used in the HRP/TMB colorimetric assay.

Mentions: 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).


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

Güven A, Fioroni M, Hauer B, Schwaneberg U - J Nanobiotechnology (2010)

Schematic representation of functionalized liposome system. The FhuA Δ1-160 channel protein embedded in the liposomal lipid membrane (left panel) employed as reduction triggered gateway for the in/out diffusion of TMB and hydrogen peroxide (right panel) used in the HRP/TMB colorimetric assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2909997&req=5

Figure 1: Schematic representation of functionalized liposome system. The FhuA Δ1-160 channel protein embedded in the liposomal lipid membrane (left panel) employed as reduction triggered gateway for the in/out diffusion of TMB and hydrogen peroxide (right panel) used in the HRP/TMB colorimetric assay.
Mentions: 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).

Bottom Line: Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Delta1-160; Onaca et al., 2008).Pyridyl labeling of K556 reduces TMB translocation to 16 [nM]/s reaching nearly background levels in liposomes (13 [nM]/s).A FhuA Delta1-160 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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany. u.schwaneberg@biotec.rwth-aachen.de.

ABSTRACT

Background: Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Delta1-160; Onaca et al., 2008). Compound fluxes within the FhuA Delta1-160 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.

Results: In total, 12 FhuA Delta1-160 variants were generated to gain insights on sterically controlled compound fluxes: Subset A) six FhuA Delta1-160 variants in which one of the six lysines in the interior of FhuA Delta1-160 was substituted to alanine and Subset B) six FhuA Delta1-160 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 Delta1-160 (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 Delta1-160 and therefore well suited for controlling compound fluxes through steric hindrance.

Conclusions: A FhuA Delta1-160 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 Delta1-160 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.

No MeSH data available.