Structural and functional characterization of a synthetically modified OmpG

Große, Wolfgang; Reiß, Philipp; Reitz, Simon; Cebi, Menekse; Lübben, Wolger; Koert, Ulrich; Essen, Lars‐Oliver

doi:10.1016/j.bmc.2010.03.044

Cited by 9 publications

(14 citation statements)

References 22 publications

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“…14a ). An interesting picture is obtained when superimposing all available X-ray structures 7 , 8 , 10 , 27 , 28 4CTD (loop 6 deletion), 2IWW, 2IWV, 2P1C, 2X9K, 2WVP (cysteine mutant synthetically modified). In this superposition, loops 3, 4, and 5 adopt very similar positions, and loops 1, 2, 6, and 7 diverge considerably, although much less so than in the NMR structures (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This contrasts with the consensus X-ray structures, in which the barrel is much longer and consists of a regular, cylindrical β-sheet. However, the superposition of related X-ray structures 7 , 8 , 10 , 27 , 28 (Supplementary Fig. 14b ) clearly shows that loops 1, 2, 6, and 7 have a degree of conformational flexibility, while loops 3, 4, and 5 look very similar, and are hence more rigid, perhaps due to restraints by interactions within a protomer or in the crystal lattice.…”

Section: Discussionmentioning

confidence: 99%

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Structure of outer membrane protein G in lipid bilayers

et al. 2017

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β-barrel proteins mediate nutrient uptake in bacteria and serve vital functions in cell signaling and adhesion. For the 14-strand outer membrane protein G of Escherichia coli, opening and closing is pH-dependent. Different roles of the extracellular loops in this process were proposed, and X-ray and solution NMR studies were divergent. Here, we report the structure of outer membrane protein G investigated in bilayers of E. coli lipid extracts by magic-angle-spinning NMR. In total, 1847 inter-residue 1H–1H and 13C–13C distance restraints, 256 torsion angles, but no hydrogen bond restraints are used to calculate the structure. The length of β-strands is found to vary beyond the membrane boundary, with strands 6–8 being the longest and the extracellular loops 3 and 4 well ordered. The site of barrel closure at strands 1 and 14 is more disordered than most remaining strands, with the flexibility decreasing toward loops 3 and 4. Loop 4 presents a well-defined helix.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Structure of outer membrane protein G in lipid bilayers

et al. 2017

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“…Single channel conductance recordings were performed using the Black Lipid Membrane (BLM) technique [18], [19], [20], [21], [22], [23]. A sample of n -decane-solubilized diphytanoyl phosphatidylcholine membrane mixture (25 mg/mL) was painted over a 200 µm circular hole, separating the chamber compartments (polystyrene cuvette: CP2A, bilayer chamber: BCH-22A, Warner Instruments ) filled equally with BLM buffer [10 mM Tris/HCl (pH 7.4), 1 M KCl, 5 mM CaCl 2 ].…”

Section: Methodsmentioning

confidence: 99%

Flexibility of the N-Terminal mVDAC1 Segment Controls the Channel’s Gating Behavior

et al. 2012

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Since the solution of the molecular structures of members of the voltage dependent anion channels (VDACs), the N-terminal α-helix has been the main focus of attention, since its strategic location, in combination with its putative conformational flexibility, could define or control the channel’s gating characteristics. Through engineering of two double-cysteine mVDAC1 variants we achieved fixing of the N-terminal segment at the bottom and midpoint of the pore. Whilst cross-linking at the midpoint resulted in the channel remaining constitutively open, cross-linking at the base resulted in an “asymmetric” gating behavior, with closure only at one electric field´s orientation depending on the channel’s orientation in the lipid bilayer. Additionally, and while the native channel adopts several well-defined closed states (S1 and S2), the cross-linked variants showed upon closure a clear preference for the S2 state. With native-channel characteristics restored following reduction of the cysteines, it is evident that the conformational flexibility of the N-terminal segment plays indeed a major part in the control of the channel’s gating behavior.

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“…OmpG from E. coli , however, has recently attracted significant attention because of its monomeric nature and the ease with which it can be modified. For engineering, OmpG is a promising template, because it forms a robust 14-stranded β-barrel with a wide, nonselective pore, which exhibits voltage- and pH-dependent gating caused by loop-driven transitions. − Current strategies for OmpG aim to deconstruct the pore or narrow the lumen permeability by introducing modulators at single or double attachment sites to transform it into a more ion- or substrate-specific porin.…”

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confidence: 99%

Structure-Based Engineering of a Minimal Porin Reveals Loop-Independent Channel Closure

et al. 2014

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Porins, like outer membrane protein G (OmpG) of Escherichia coli, are ideal templates among ion channels for protein and chemical engineering because of their robustness and simple architecture. OmpG shows fast transitions between open and closed states, which were attributed to loop 6 (L6). As flickering limits single-channel-based applications, we pruned L6 by either 8 or 12 amino acids. While the open probabilities of both L6 variants resemble that of native OmpG, their gating frequencies were reduced by 63 and 81%, respectively. Using the 3.2 Å structure of the shorter L6 variant in the open state, we engineered a minimal porin (220 amino acids), where all remaining extramembranous loops were truncated. Unexpectedly, this minimized porin still exhibited gating, but it was 5-fold less frequent than in OmpG. The residual gating of the minimal pore is hence independent of L6 rearrangements and involves narrowing of the ion conductance pathway most probably driven by global stretching-flexing deformations of the membrane-embedded β-barrel.

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Structural and functional characterization of a synthetically modified OmpG

Cited by 9 publications

References 22 publications

Structure of outer membrane protein G in lipid bilayers

Structure of outer membrane protein G in lipid bilayers

Flexibility of the N-Terminal mVDAC1 Segment Controls the Channel’s Gating Behavior

Structure-Based Engineering of a Minimal Porin Reveals Loop-Independent Channel Closure

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