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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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Φ-Value analysis of HT PagP. ΦF-values determined from kinetic analysis of HT PagP variants are mapped onto a ribbon diagram (left) and a topology model (right). Regions with ΦF-values close to 0 are shown in red, regions with ΦF-values close to 1 are shown in blue, intermediate ΦF-values are shown in purple, ΦF-values less than 1 are shown in orange and undetermined ΦF-values are grey. Reproduced with permission from [157].
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f0045: Φ-Value analysis of HT PagP. ΦF-values determined from kinetic analysis of HT PagP variants are mapped onto a ribbon diagram (left) and a topology model (right). Regions with ΦF-values close to 0 are shown in red, regions with ΦF-values close to 1 are shown in blue, intermediate ΦF-values are shown in purple, ΦF-values less than 1 are shown in orange and undetermined ΦF-values are grey. Reproduced with permission from [157].

Mentions: By systematically varying the protein concentration and studying the folding of HT PagP under a range of lipid-to-protein ratios (LPRs), conditions were established under which the HT PagP unfolding transition is completely reversible in diC12:0PC LUVs [157]. Equilibrium stability studies and kinetic chevron plot analysis of HT PagP (un)folding revealed that the protein folds via a two-state mechanism over the range of urea concentrations studied (7.8–10 M). A Φ-value analysis was then undertaken for HT PagP using point mutants of 19 residues spread throughout the protein structure [157]. These experiments provided the first insights into the structural features of a transition state for OMP folding, suggesting a polarised transition state in which the N-terminal half of the protein remains largely unstructured, whilst the C-terminal half of the protein is native-like (Fig. 9) [157]. Interestingly, two negative Φ-values were observed, providing evidence for stabilisation of the transition state by non-native interactions [157]. The resulting mechanism of tilted insertion is consistent with the concerted folding and insertion suggested for OmpA [143,144,170]. It remains to be seen whether this mechanism is observed for other OMPs.


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)

Φ-Value analysis of HT PagP. ΦF-values determined from kinetic analysis of HT PagP variants are mapped onto a ribbon diagram (left) and a topology model (right). Regions with ΦF-values close to 0 are shown in red, regions with ΦF-values close to 1 are shown in blue, intermediate ΦF-values are shown in purple, ΦF-values less than 1 are shown in orange and undetermined ΦF-values are grey. Reproduced with permission from [157].
© Copyright Policy
Related In: Results  -  Collection

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

f0045: Φ-Value analysis of HT PagP. ΦF-values determined from kinetic analysis of HT PagP variants are mapped onto a ribbon diagram (left) and a topology model (right). Regions with ΦF-values close to 0 are shown in red, regions with ΦF-values close to 1 are shown in blue, intermediate ΦF-values are shown in purple, ΦF-values less than 1 are shown in orange and undetermined ΦF-values are grey. Reproduced with permission from [157].
Mentions: By systematically varying the protein concentration and studying the folding of HT PagP under a range of lipid-to-protein ratios (LPRs), conditions were established under which the HT PagP unfolding transition is completely reversible in diC12:0PC LUVs [157]. Equilibrium stability studies and kinetic chevron plot analysis of HT PagP (un)folding revealed that the protein folds via a two-state mechanism over the range of urea concentrations studied (7.8–10 M). A Φ-value analysis was then undertaken for HT PagP using point mutants of 19 residues spread throughout the protein structure [157]. These experiments provided the first insights into the structural features of a transition state for OMP folding, suggesting a polarised transition state in which the N-terminal half of the protein remains largely unstructured, whilst the C-terminal half of the protein is native-like (Fig. 9) [157]. Interestingly, two negative Φ-values were observed, providing evidence for stabilisation of the transition state by non-native interactions [157]. The resulting mechanism of tilted insertion is consistent with the concerted folding and insertion suggested for OmpA [143,144,170]. It remains to be seen whether this mechanism is observed for other OMPs.

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