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

Journal of Nanobiotechnology

Research

Bernhard Hauer¹  Ulrich Schwaneberg²  Marco Fioroni²  Arcan Güven³ 

[1] Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, D-70569, Stuttgart, Germany;Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany;School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759, Bremen, Germany;Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany;

关键词: Calcein;    MscL;    Outer Membrane Fraction;    Channel Interior;    Thin Lipid Film;   

DOI  :  10.1186/1477-3155-8-14

 received in 2010-03-16, accepted in 2010-06-25,  发布年份 2010

来源: Springer

【 摘 要 】

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.

【 授权许可】

Unknown   
© Güven et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.