On the Function and Structure of Synthetically Modified Porins

Reitz, Simon; Cebi, Menekse; Reiß, Philipp; Studnik, Gregor; Linne, Uwe; Koert, Ulrich; Essen, Lars‐Oliver

doi:10.1002/anie.200900457

Cited by 22 publications

(19 citation statements)

References 23 publications

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“…However, since divalent cations are known to induce pKa shifts of key acidic OmpF residues and affect ion-selectivity of the channel (Queralt-Martin et al, 2011), a reduced concentration of MgCl 2 (200 mM) was used, as previously reported (Reitz et al, 2009). Crystals of E. coli OmpF protein were obtained at 20 °C in a hanging drop (VDX plates, Hampton Research) with the reservoir solution, 100 mM sodium cacodylate, 200 mM MgCl 2 , 50% (w/v) PEG 200, pH 6.5 mixed in a 1:1 ratio with protein solution, 20 mM Tris-HCl, 0.1 M NaCl, 0.8% octyl-POE, pH 8.0; OmpF concentration 5–8 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

The Binding of Antibiotics in OmpF Porin

2013

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The structure of OmpF porin in complex with three common antibiotics (zwitterionic ampicillin, anionic ertapenem, and di-anionic carbenicillin) was determined using X-ray crystallography. The three antibiotics are found to bind within the extracellular and periplasmic pore vestibules, away from the narrow OmpF constriction zone. Using the X-ray structures as a starting point, non-equilibrium MD simulations with an applied membrane voltage show that ionic current through the OmpF channel is blocked with bound ampicillin, but not with bound carbenicillin. The susceptibility of E. coli expressing OmpF mutants to ampicillin and carbenicillin was also experimentally characterized using microbiological assays. These results show that general diffusion by OmpF porins allows for transfer of molecules with varied charged states and give new insights into the design of more efficient antibiotics. A better understanding of this mechanism will shed light on nature's way of devising channels able to enhance the transport of molecules through membranes.

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Section: Methodsmentioning

confidence: 99%

The Binding of Antibiotics in OmpF Porin

2013

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“…Bacterial outer membrane proteins (and specifically porins) offer a good starting point for biomimetic design. As a consequence of their β-barrel architecture, they are stable and readily mutatable bionanopores, , offering a range of functionalities . Porin selectivity seems to largely reside in the protein side chains rather than in the backbone as is the case in K channels and in aquaporins .…”

Section: Introductionmentioning

confidence: 99%

Biomimetic Design of a Brush-Like Nanopore: Simulation Studies

2011

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Combining a high degree of selectivity and nanoscale dimensions, biological pores are attractive potential components for nanotechnology devices and applications. Biomimetic design will facilitate production of stable synthetic nanopores with defined functionality. Bacterial porins offer a good source of possible templates for such nanopores as they form stable, selective pores in lipid bilayers. Molecular dynamics simulations have been used to design simple model nanopores with permeation free energy profiles that can be made to mimic a template protein, the OprP porin, which forms pores selective for anions. In particular, we explored the effects of varying the nature of pore-lining groups on free energy profiles for phosphate and chloride ions along the pore axis and the total charge of the permeation pathway of the pore. Cationic side chains lining the model nanopore are required to model the local detail of the OprP permeation landscape, whereas the total charge contributes to its magnitude. These studies indicate that a locally accurate biomimetic design has captured the essentials of the structure/function relationship of the parent protein.

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“…Secondly, the alternative reaction in the denatured state using 6 M urea was performed before the hybrid dodecin was refolded, to counter problems with the size of the chemical compound which may prevent its accommodation within the central cavity. The chemical modification of proteins with the used dansyl compound (N-(2-ethyl-iodo-acetamide)-dansyl) 17 (see ESIw) was applicable in the folded and unfolded state of dodecin. The success of the almost stoichiometric modification reactions was examined by mass spectrometry and SDS-PAGE (see ESIw 2.1 and 2.3), where the intrinsic fluorescence of dansyl under UV light (l exc = 325 nm) was used for detection (Fig.…”

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confidence: 99%