In vitro folding and oligomerization of a membrane protein. Transition of bacterial porin from random coil to native conformation.

Eiselé, Jean Luc; Rosenbusch, J P

doi:10.1016/s0021-9258(18)86933-2

Cited by 115 publications

(28 citation statements)

References 31 publications

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“…Two distinct changes are evident upon folding: a trough appears at 216 nm indicative of β-sheet structure, and a peak appears at 230 nm. The observed β-sheet intensity of −5000 deg cm 2 dmol –1 residue –1 is highly consistent with all previous CD data on OMPs. ,,,,,− Although not as common, CD bands in the 230 nm region of the spectrum have previously been observed in a number of soluble proteins and have been attributed to aromatic residues that interact to give rise to exciton couplets in the spectra. − Similarly, the OMP PagP exhibits a peak at 232 nm, which has been attributed to the interaction of a specific pair of aromatic residues brought into close proximity across the interior of the folded β-barrel . OmpA 171 contains a large number of aromatic residues, and we propose that a similar interaction occurs, producing a peak of 700 deg cm 2 dmol –1 residue –1 at 230 nm.…”

Section: Resultssupporting

confidence: 86%

Novel Kinetic Intermediates Populated along the Folding Pathway of the Transmembrane β-Barrel OmpA

Danoff

Fleming

2016

Biochemistry

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We examined the folding of the β-barrel membrane protein OmpA from E. coli. Although previous studies identified several intermediate states followed by a concerted translocation mechanism across the bilayer, some aspects of the pathway were still unclear including the extent of secondary structure formation in the intermediate states and how the mechanism gave rise to multiple exponential phases in the folding kinetics. We addressed these questions by investigating the folding kinetics of the OmpA transmembrane β-barrel domain into a range of bilayer thicknesses, allowing us to observe different regions of the folding pathway. The fastest folding into the thinnest bilayers provided information on the later stages of the process, and the slowest folding into thicker bilayers revealed early kinetic steps. Folding was monitored using SDS-PAGE and CD spectroscopy, which provide complementary information about tertiary and secondary structure formation. We globally fit the folding data to kinetic schemes and found that the same core pathway was followed in all lipid conditions. We propose a multi-step folding mechanism for OmpA that includes unstructured surface-adsorbed states converting through a partially inserted state with substantial β-sheet structure to the final natively inserted barrel. Kinetic models show that all steps of the main folding pathway are accelerated by membrane defects that occur as a result of thinning the bilayer or incubation of lipids at the phase transition temperature. In addition to suppressing off-pathway states, BAM-catalyzed folding in vivo could accelerate any or all of these main folding steps to ensure efficient OMP biogenesis in vivo.

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Section: Resultssupporting

confidence: 86%

Novel Kinetic Intermediates Populated along the Folding Pathway of the Transmembrane β-Barrel OmpA

Danoff

Fleming

2016

Biochemistry

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“…Subsequently, the addition of sterol to purified porin led to a 10-fold increase of the channel-forming activity for porin from D. discoideum and to a smaller extent of that of Paramecium and rat liver. This result indicates that the stability of the channel-forming complex is much smaller for mitochondrial porins as compared to that of bacterial porins, which easily withstand even treatment with SDS (Schindler & Rosenbusch, 1978;Eisele & Rosenbusch, 1990).…”

Section: Discussionmentioning

confidence: 94%

Role of Sterols in the Functional Reconstitution of Water-Soluble Mitochondrial Porins from Different Organisms

Popp¹,

Schmid²,

Benz³

1995

Biochemistry

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Experiments were performed on lipid bilayer membranes with water-soluble mitochondrial porins from different eukaryotic organisms, such as Dictyostelium discoideum, Paramecium, and rat liver, to study the requirements of functional reconstitution of the porins. The water-soluble porins lost their associated lipids and sterols and are unable to form channels in lipid bilayer membranes. We demonstrate that the water-soluble porins regain their channel-forming ability after preincubation of the polypeptides with sterols in the presence of detergents. Mitochondrial porin from Dictyostelium discoideum maintained after this procedure its original properties, in particular the voltage dependence. Water-soluble mitochondrial porins from Paramecium tetraurelia and from rat liver were also activated upon preincubation with different sterols in detergent but showed voltage-dependences that were different from those of detergent-purified porins. Furthermore, the voltage dependence depended on the sterol used for preincubation. Interestingly, the preincubation with sterols can likewise be used to activate detergent-purified mitochondrial porins that may have lost associated sterol during isolation and purification procedures.

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“…As a result, if OMPs are retained in the cytoplasm, they are unable to fold into their native conformation and then aggregate. These OMPs can be extracted from aggregated cytoplasmic inclusion bodies with denaturing agents, and then refolded in vitro in the presence of detergents Eisele & Rosenbusch, 1990). The refolding efficiency is generally enhanced by the addition of chaperones and LPS (Bulieris et al, 2003).…”

Section: Discussionmentioning

confidence: 99%

Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli

et al. 2009

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TolC is a multifunctional outer-membrane protein (OMP) of Escherichia coli that folds into a unique α/β-barrel structure. Previous studies have shown that unlike the biogenesis of β-barrel OMPs, such as porins, TolC assembles independently from known periplasmic folding factors. Yet, the assembly of TolC, like that of β-barrel OMPs, is dependent on BamA and BamD, two essential components of the β-barrel OMP assembly machinery. We have investigated the folding properties and cellular trafficking of a TolC derivative that lacks the entire signal sequence (TolCΔ2–22). A significant amount of TolCΔ2–22 was found to be soluble in the cytoplasm, and a fraction of it folded and trimerized into a conformation similar to that of the normal outer membrane-localized TolC protein. Some TolCΔ2–22 was found to associate with membranes, but failed to assume a wild-type-like folded conformation. The null phenotype of TolCΔ2–22 was exploited to isolate suppressor mutations, the majority of which mapped in secY. In the secY suppressor background, TolCΔ2–22 resumed normal function and folded like wild-type TolC. Proper membrane insertion could not be achieved upon in vitro incubation of cytoplasmically folded TolCΔ2–22 with purified outer membrane vesicles, showing that even though TolC is intrinsically capable of folding and trimerization, for successful integration into the outer membrane these events need to be tightly coupled to the insertion process, which is mediated by the Bam machinery. Genetic and biochemical data attribute the unique folding and assembly pathways of TolC to its large soluble α-helical domain.

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In vitro folding and oligomerization of a membrane protein. Transition of bacterial porin from random coil to native conformation.

Cited by 115 publications

References 31 publications

Novel Kinetic Intermediates Populated along the Folding Pathway of the Transmembrane β-Barrel OmpA

Novel Kinetic Intermediates Populated along the Folding Pathway of the Transmembrane β-Barrel OmpA

Role of Sterols in the Functional Reconstitution of Water-Soluble Mitochondrial Porins from Different Organisms

Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli

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