In vitro trimerization of outer membrane protein PhoE

Detergent‐Induced Folding of the Outer‐Membrane Protein PhoE, a Pore Protein Induced by Phosphate Limitation

“…However, the trimers obtained were apparently not inserted into the outer membranes, since they remained in the supernatant after pelleting of the membranes [4]. Furthermore, in contrast to PhoE trimers synthesized in vivu, the trimers obtained in the in vitro system seemed to be sensitive to proteases [4]. This picture is now drastically changed by the results obtained in the present study.…”

“…Cell envelopes stimulated the rate of trimerization possibly by serving as 'concentration points', increasing the local concentration of the PhoE molecules. Thirdly, the observation that the in vitro detected PhoE trimers apparently remained in the supernatant after pelleting of the outer membranes [4] can now easily be explained. The trimers detected were most likely only formed in the supernatant fraction during the immunoprecipitation in the presence of Triton X-100, i.e.…”

Detergent‐Induced Folding of the Outer‐Membrane Protein PhoE, a Pore Protein Induced by Phosphate Limitation

“…However, the trimers obtained were apparently not inserted into the outer membranes, since they remained in the supernatant after pelleting of the membranes [4]. Furthermore, in contrast to PhoE trimers synthesized in vivu, the trimers obtained in the in vitro system seemed to be sensitive to proteases [4]. This picture is now drastically changed by the results obtained in the present study.…”

“…Cell envelopes stimulated the rate of trimerization possibly by serving as 'concentration points', increasing the local concentration of the PhoE molecules. Thirdly, the observation that the in vitro detected PhoE trimers apparently remained in the supernatant after pelleting of the outer membranes [4] can now easily be explained. The trimers detected were most likely only formed in the supernatant fraction during the immunoprecipitation in the presence of Triton X-100, i.e.…”

Lipopolysaccharides and divalent cations are involved in the formation of an assembly‐competent intermediate of outer‐membrane protein PhoE of E.coli.

“…for the folding into a folded monomer. The conditions for the formation of the assembly-competent folded monomer require very low amounts of the non-ionic detergent TX-100 (0.015%), LPS and divalent cations, conditions that were previously not met (de Cock et al, 1990a(de Cock et al, ,b, 1996Van Gelder et al, 1994). Previously, we demonstrated that the detergent is required for the formation of an assembly-competent intermediate of PhoE protein with a short half-life (de Cock et al, 1996).…”

In Vitro Insertion and Assembly of Outer Membrane Protein PhoE of Escherichia coli K-12 into the Outer Membrane

“…In this latter system, the presence of TX-100 was not required for trimerization. In contrast, in vitro synthesized PhoE protein was not assembled into a trypsin-resistant configuration when incubated with OMs in the absence of TX-100 (16). Here, we demonstrate that low amounts of TX-100 strongly stimulate the assembly of PhoE into purified OMs, and we investigated the role of the detergent and of LPS in this process in more detail.…”

Detergent‐Induced Folding of the Outer‐Membrane Protein PhoE, a Pore Protein Induced by Phosphate Limitation

“…However, the trimers obtained were apparently not inserted into the outer membranes, since they remained in the supernatant after pelleting of the membranes [4]. Furthermore, in contrast to PhoE trimers synthesized in vivu, the trimers obtained in the in vitro system seemed to be sensitive to proteases [4]. This picture is now drastically changed by the results obtained in the present study.…”

“…Cell envelopes stimulated the rate of trimerization possibly by serving as 'concentration points', increasing the local concentration of the PhoE molecules. Thirdly, the observation that the in vitro detected PhoE trimers apparently remained in the supernatant after pelleting of the outer membranes [4] can now easily be explained. The trimers detected were most likely only formed in the supernatant fraction during the immunoprecipitation in the presence of Triton X-100, i.e.…”

Lipopolysaccharides and divalent cations are involved in the formation of an assembly‐competent intermediate of outer‐membrane protein PhoE of E.coli.

“…for the folding into a folded monomer. The conditions for the formation of the assembly-competent folded monomer require very low amounts of the non-ionic detergent TX-100 (0.015%), LPS and divalent cations, conditions that were previously not met (de Cock et al, 1990a(de Cock et al, ,b, 1996Van Gelder et al, 1994). Previously, we demonstrated that the detergent is required for the formation of an assembly-competent intermediate of PhoE protein with a short half-life (de Cock et al, 1996).…”

In Vitro Insertion and Assembly of Outer Membrane Protein PhoE of Escherichia coli K-12 into the Outer Membrane

“…In this latter system, the presence of TX-100 was not required for trimerization. In contrast, in vitro synthesized PhoE protein was not assembled into a trypsin-resistant configuration when incubated with OMs in the absence of TX-100 (16). Here, we demonstrate that low amounts of TX-100 strongly stimulate the assembly of PhoE into purified OMs, and we investigated the role of the detergent and of LPS in this process in more detail.…”

Detergent‐Induced Folding of the Outer‐Membrane Protein PhoE, a Pore Protein Induced by Phosphate Limitation

“…However, the trimers obtained were apparently not inserted into the outer membranes, since they remained in the supernatant after pelleting of the membranes [4]. Furthermore, in contrast to PhoE trimers synthesized in vivu, the trimers obtained in the in vitro system seemed to be sensitive to proteases [4]. This picture is now drastically changed by the results obtained in the present study.…”

“…Cell envelopes stimulated the rate of trimerization possibly by serving as 'concentration points', increasing the local concentration of the PhoE molecules. Thirdly, the observation that the in vitro detected PhoE trimers apparently remained in the supernatant after pelleting of the outer membranes [4] can now easily be explained. The trimers detected were most likely only formed in the supernatant fraction during the immunoprecipitation in the presence of Triton X-100, i.e.…”

Lipopolysaccharides and divalent cations are involved in the formation of an assembly‐competent intermediate of outer‐membrane protein PhoE of E.coli.

“…for the folding into a folded monomer. The conditions for the formation of the assembly-competent folded monomer require very low amounts of the non-ionic detergent TX-100 (0.015%), LPS and divalent cations, conditions that were previously not met (de Cock et al, 1990a(de Cock et al, ,b, 1996Van Gelder et al, 1994). Previously, we demonstrated that the detergent is required for the formation of an assembly-competent intermediate of PhoE protein with a short half-life (de Cock et al, 1996).…”

In Vitro Insertion and Assembly of Outer Membrane Protein PhoE of Escherichia coli K-12 into the Outer Membrane

“…In this latter system, the presence of TX-100 was not required for trimerization. In contrast, in vitro synthesized PhoE protein was not assembled into a trypsin-resistant configuration when incubated with OMs in the absence of TX-100 (16). Here, we demonstrate that low amounts of TX-100 strongly stimulate the assembly of PhoE into purified OMs, and we investigated the role of the detergent and of LPS in this process in more detail.…”