Towards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case

Roussel, G.; Perpète, Éric A.; Matagne, André; Tinti, Emmanuel; Michaux, Catherine

doi:10.1002/bit.24722

Cited by 19 publications

(24 citation statements)

References 59 publications

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“…From Figure , we can state that while MPD alone does not change the peptide structure, the co‐solvent in concentration ranging from 1.5 M to 2.5 M totally prevents the denaturation of both peptides by SDS micelles, even after a 1‐h incubation. On the other hand, MPD is no more able to protect the peptides against unfolding when its concentration falls below 0.75 M. This is in line with previous observations establishing that a high concentration (1–2 M) of MPD is needed to change the SDS denaturing properties.…”

Section: Resultssupporting

confidence: 89%

“…The observed trends are similar for TrpZip and TrpCage: up to 1000 mM NaCl, adding salt has a beneficial effect on the efficiency index whereas further increasing the NaCl concentration reduces the process' efficacy. This is completely in agreement with previous results showing that the membrane protein Omp2a renaturation is gradually favoured by increasing the salt concentration up to a maximum of 400 mM NaCl [9].…”

Section: Ionic Strength Effectsupporting

confidence: 93%

“…Eventually, the position of alcohol group turns out to be crucial. Noteworthily, these results strongly corroborate the trends that were also observed for the membrane protein Pagp [9] (Table S2).…”

Section: Efficiency Of Mpd and Sds Analoguessupporting

confidence: 88%

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Unravelling the mechanisms of a protein refolding process based on the association of detergents and co-solvents

et al. 2016

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A new technique associating the detergent Sodium Dodecyl Sulphate (SDS) and an alcohol-type co-solvent has been set up, showing an unexpected efficiency to refold several types of soluble or membrane proteins. The present contribution deepens the fundamental knowledge on the phenomena underlying this process, considering the refolding of two model peptides featuring the main protein secondary structures: α-helix and β-sheet. Their refolding was monitored by fluorescence and circular dichroism, and it turns out that: (i) 100% recovery of the folded structure is observed for both peptides, (ii) the highest the SDS concentration, the more co-solvent to be added to recover the peptides' native structures, (iii) a high alcohol concentration is required to alter the SDS denaturing properties, (iv) the co-solvent performance relies on its specific lipophilic-hydrophilic balanced character, (v) the size of the micelle formed by the detergent does not enter the process critical parameters, and (vi) increasing the salt concentration up to 1 M NaCl has a beneficial impact on the process efficiency. These mechanistic aspects will help us to improve the method and extend its application. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

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Section: Resultssupporting

confidence: 89%

Section: Ionic Strength Effectsupporting

confidence: 93%

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Unravelling the mechanisms of a protein refolding process based on the association of detergents and co-solvents

et al. 2016

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“…Inclusion bodies of PsOep23 were solubilized in Buffer A (10 mM sodium phosphate pH 7.5, 120 mM SDS) containing 150 mM or 400 mM NaF and subsequently diluted 1:2 in the same buffers containing 3 M 2-methyl-2,4-pentandiol instead of SDS (buffer condition 1 and 2, respectively, adapted from [ 35 ]). PsOep23 was refolded for 5 days at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Oep23 forms an ion channel in the chloroplast outer envelope

et al. 2015

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BackgroundMetabolite, ion and protein translocation into chloroplasts occurs across two membranes, the inner and the outer envelope. Solute and metabolite channels fulfill very important functions in integrating the organelles into the metabolic network of the cell. However so far only a few have been identified. Here we describe the identification and the characterization of the outer envelope protein of 23 kDa, Oep23 from garden pea.ResultsOep23 is found in the entire plant lineage from green algae to flowering plants. It is expressed in all organs and developmental states tested so far. The reconstituted recombinant protein Oep23 from pea forms a high conductance ion channel with a maximal conductance in the fully open state of 466 ± 14pS at a holding potential of +100 mV (in 250 mM KCl). The Oep23 channel is cation selective (PK+ : PCl- = 15 : 1) with a voltage dependent open probability of maximal Vmem = 0 mV.ConclusionThe data indicate that the Oep23 activity represents a single channel unit and does not assemble into a multiple pore complex like bacterial type porins or mitochondrial voltage dependent anion channel. Thus, Oep23 represents a new member of ion channels in the outer envelope of chloroplasts involved in solute exchange.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0445-1) contains supplementary material, which is available to authorized users.

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“…65. Therefore, IBs (see above) were solubilized in SDS buffer (120 mM SDS, 10 mM NaP i , pH 7.5, 200 mM NaF) by rotation for 1 h at 20°C.…”

Section: Single Channel Recordings From Planar Lipid Bilayers Andmentioning

confidence: 99%

OEP40, a Regulated Glucose-permeable β-Barrel Solute Channel in the Chloroplast Outer Envelope Membrane

Harsman

Schock

Hemmis

et al. 2016

Journal of Biological Chemistry

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Chloroplasts and mitochondria are unique endosymbiotic cellular organelles surrounded by two membranes. Essential metabolic networking between these compartments and their hosting cells requires the exchange of a large number of biochemical pathway intermediates in a directed and coordinated fashion across their inner and outer envelope membranes. Here, we describe the identification and functional characterization of a highly specific, regulated solute channel in the outer envelope of chloroplasts, named OEP40. Loss of OEP40 function in Arabidopsis thaliana results in early flowering under cold temperature. The reconstituted recombinant OEP40 protein forms a high conductance ␤-barrel ion channel with subconductant states in planar lipid bilayers. The OEP40 channel is slightly cation-selective P K؉ /P Cl؊ ≈ 4:1 and rectifying (ı ᠬ/ı ᠭ Х 2) with a slope conductance of Ḡ max Х 690 picosiemens. The OEP40 channel has a restriction zone diameter of Х1.4 nm and is permeable for glucose, glucose 1-phosphate and glucose 6-phosphate, but not for maltose. Moreover, channel properties are regulated by trehalose 6-phosphate, which cannot permeate. Altogether, our results indicate that OEP40 is a "glucose-gate" in the outer envelope membrane of chloroplasts, facilitating selective metabolite exchange between chloroplasts and the surrounding cell.The plant cell is highly compartmentalized, containing at least seven different types of organelles that are involved in metabolism and cellular maintenance. In many cases, metabolic pathways and other cellular processes are not confined to one organelle but require the cooperation of several of them, e.g. in protein secretion or photorespiration. Metabolic networking between compartments thus requires directed and coordinated exchange of biochemical pathway intermediates across organellar membranes (1). Chloroplasts and mitochondria are unique endosymbiotic cellular organelles, which like their prokaryotic ancestors are surrounded by two membranes. The inner membrane of both organelles is derived from the bacterial plasma membrane. Surprisingly, also the outer membrane largely originated from and still resembles the outer membrane of the Gram-negative-like bacterial endosymbiont (2). In the inner chloroplast envelope membrane (IE), 5 numerous metabolite transporter proteins were identified and characterized to large detail with respect to their physiological impact and molecular mechanisms (3). These transporters are hydrophobic, polytopic, and mainly ␣-helical membrane proteins facilitating the exchange of metabolic precursors, intermediates, and final products between plastids and the cytoplasm. In contrast, the characteristic channels of the outer membranes in bacteria, chloroplasts as well as mitochondria, span the membrane in the form of ␤-strands that are organized to form a barrel-like pore structure (4). Proteins sharing this three-dimensional arrangement were implied in a variety of functions, including protein import (5). In addition, solute pores like the voltage-dependen...

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Towards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case

Cited by 19 publications

References 59 publications

Unravelling the mechanisms of a protein refolding process based on the association of detergents and co-solvents

Unravelling the mechanisms of a protein refolding process based on the association of detergents and co-solvents

Oep23 forms an ion channel in the chloroplast outer envelope

OEP40, a Regulated Glucose-permeable β-Barrel Solute Channel in the Chloroplast Outer Envelope Membrane

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