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Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro and in vivo: what have we learned to date?

McMorran LM, Brockwell DJ, Radford SE - Arch. Biochem. Biophys. (2014)

Bottom Line: Using the panoply of methods developed for studies of the folding of water-soluble proteins.This review summarises current knowledge of the mechanisms of outer membrane protein biogenesis and folding into lipid bilayers in vivo and in vitro and discusses the experimental techniques utilised to gain this information.The emerging knowledge is beginning to allow comparisons to be made between the folding of membrane proteins with current understanding of the mechanisms of folding of water-soluble proteins.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

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Related in: MedlinePlus

Schematic of the effects of Skp and SurA on the refolding of PagP. SurA and PagP do not interact stably under the conditions of the refolding assay. Skp readily interacts with PagP, retarding the PagP folding rate into zwitterionic liposomes, but accelerating the folding rate of PagP into negatively charged liposomes in a manner dependent on the ionic strength of the buffer. Additionally, the holdase activity of Skp was demonstrated by its ability to rescue the folding and membrane insertion of HT PagP under conditions which strongly favour aggregation of this construct. This figure was adapted from [92] with permission from Elsevier, © 2013.
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f0050: Schematic of the effects of Skp and SurA on the refolding of PagP. SurA and PagP do not interact stably under the conditions of the refolding assay. Skp readily interacts with PagP, retarding the PagP folding rate into zwitterionic liposomes, but accelerating the folding rate of PagP into negatively charged liposomes in a manner dependent on the ionic strength of the buffer. Additionally, the holdase activity of Skp was demonstrated by its ability to rescue the folding and membrane insertion of HT PagP under conditions which strongly favour aggregation of this construct. This figure was adapted from [92] with permission from Elsevier, © 2013.

Mentions: More recently, kinetic analysis of an untagged variant of PagP has been undertaken in the presence of SurA and Skp [92]. This PagP construct, initially reported by Burgess et al. [200], was chosen for folding assays in the presence of soluble chaperones as it had been reported to be folding competent in urea concentrations as low as 1 M, in stark contrast with the high urea concentrations required for efficient folding of HT PagP [200,211]. Interestingly, however, folding of PagP is not fully reversible under conditions which promote reversible folding of HT PagP, again highlighting the difficulty in generating OMPs suitable for equilibrium denaturation studies [92]. Nonetheless, the folding kinetics of PagP into both zwitterionic and negatively charged liposomes were investigated in the presence and absence of Skp or SurA (Fig. 10) [92]. These experiments showed that membrane composition and ionic strength of the buffer strongly influences the effect that Skp has on PagP folding, suggesting that electrostatic interactions play an important role in the mechanism of action of this chaperone [92], consistent with the previous results on Skp-mediated folding of OmpA [92,182]. SurA, however, did not affect the observed folding rates of PagP, in contrast with the results observed for OmpT refolding [135], but consistent with the view that Skp and SurA may act by distinct mechanisms in partially redundant chaperone pathways [78,105]. The ability of Skp to prevent the aggregation of HT PagP was also investigated, revealing that even under conditions in which aggregation is strongly favoured, Skp can rescue the folding and membrane insertion of HT PagP [92]. Together, these studies indicate the power of combining different methods to study the folding mechanism of an OMP and set the scope for future investigations into how OMPs fold both unassisted and assisted by folding factors in vitro and in vivo.


Mechanistic studies of the biogenesis and folding of outer membrane proteins in vitro and in vivo: what have we learned to date?

McMorran LM, Brockwell DJ, Radford SE - Arch. Biochem. Biophys. (2014)

Schematic of the effects of Skp and SurA on the refolding of PagP. SurA and PagP do not interact stably under the conditions of the refolding assay. Skp readily interacts with PagP, retarding the PagP folding rate into zwitterionic liposomes, but accelerating the folding rate of PagP into negatively charged liposomes in a manner dependent on the ionic strength of the buffer. Additionally, the holdase activity of Skp was demonstrated by its ability to rescue the folding and membrane insertion of HT PagP under conditions which strongly favour aggregation of this construct. This figure was adapted from [92] with permission from Elsevier, © 2013.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4262575&req=5

f0050: Schematic of the effects of Skp and SurA on the refolding of PagP. SurA and PagP do not interact stably under the conditions of the refolding assay. Skp readily interacts with PagP, retarding the PagP folding rate into zwitterionic liposomes, but accelerating the folding rate of PagP into negatively charged liposomes in a manner dependent on the ionic strength of the buffer. Additionally, the holdase activity of Skp was demonstrated by its ability to rescue the folding and membrane insertion of HT PagP under conditions which strongly favour aggregation of this construct. This figure was adapted from [92] with permission from Elsevier, © 2013.
Mentions: More recently, kinetic analysis of an untagged variant of PagP has been undertaken in the presence of SurA and Skp [92]. This PagP construct, initially reported by Burgess et al. [200], was chosen for folding assays in the presence of soluble chaperones as it had been reported to be folding competent in urea concentrations as low as 1 M, in stark contrast with the high urea concentrations required for efficient folding of HT PagP [200,211]. Interestingly, however, folding of PagP is not fully reversible under conditions which promote reversible folding of HT PagP, again highlighting the difficulty in generating OMPs suitable for equilibrium denaturation studies [92]. Nonetheless, the folding kinetics of PagP into both zwitterionic and negatively charged liposomes were investigated in the presence and absence of Skp or SurA (Fig. 10) [92]. These experiments showed that membrane composition and ionic strength of the buffer strongly influences the effect that Skp has on PagP folding, suggesting that electrostatic interactions play an important role in the mechanism of action of this chaperone [92], consistent with the previous results on Skp-mediated folding of OmpA [92,182]. SurA, however, did not affect the observed folding rates of PagP, in contrast with the results observed for OmpT refolding [135], but consistent with the view that Skp and SurA may act by distinct mechanisms in partially redundant chaperone pathways [78,105]. The ability of Skp to prevent the aggregation of HT PagP was also investigated, revealing that even under conditions in which aggregation is strongly favoured, Skp can rescue the folding and membrane insertion of HT PagP [92]. Together, these studies indicate the power of combining different methods to study the folding mechanism of an OMP and set the scope for future investigations into how OMPs fold both unassisted and assisted by folding factors in vitro and in vivo.

Bottom Line: Using the panoply of methods developed for studies of the folding of water-soluble proteins.This review summarises current knowledge of the mechanisms of outer membrane protein biogenesis and folding into lipid bilayers in vivo and in vitro and discusses the experimental techniques utilised to gain this information.The emerging knowledge is beginning to allow comparisons to be made between the folding of membrane proteins with current understanding of the mechanisms of folding of water-soluble proteins.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK; School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

Show MeSH
Related in: MedlinePlus