Abstract
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.
Highlights
Channel proteins like Ferric hydroxamate protein uptake component A (FhuA) can be an alternative to artificial chemically synthesized nanopores
Since the FhuA shows a nearly circular morphology [11,12], a simple regular polygonal geometry, with constrained side length given by the hydrogen bond connecting the b-sheets, can be assumed (Figure 2)
Based on this assumption the relative expected diameter increase can be calculated, as the FhuA wild type (WT) channel diameter with 22 b-sheets is known to be ~4.2 nm as deduced from crystal structure [11,12]
Summary
Channel 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. The smallest known barrel (i.e. OmpA) contains 8 transmembrane strands; due to packing constraints in the. Apart from their biological importance, one application of bacterial membrane proteins with b-barrel structure is the channel functionalization of lipid or block copolymer based membranes. The FhuA proved to be useful, due to its wide channel diameter and robustness against for instance tryptic digestion [10]
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