PMID:8855933

Citation	Kleinschmidt, JH and Tamm, LK (1996) Folding intermediates of a beta-barrel membrane protein. Kinetic evidence for a multi-step membrane insertion mechanism. Biochemistry 35:12993-3000
Abstract	The mechanism of folding and membrane insertion of integral membrane proteins, including helix bundle and beta-barrel proteins is not well understood. A key question is whether folding and insertion are coupled or separable processes. We have used the beta-barrel outer membrane protein A (OmpA) of Escherichia coli as a model to study the kinetics of folding and insertion into dioleoylphosphatidylcholine (DOPC) bilayers, as a function of temperature by gel electrophoresis, protease digestion, and fluorescence spectroscopy. OmpA was unfolded in 8 M urea solution (without detergent), and refolding and membrane insertion was initiated by rapid dilution of the urea concentration in the presence of phospholipid vesicles. In addition to the kinetically unresolved hydrophobic collapse in water, the time course of refolding of OmpA into DOPC bilayers exhibited three kinetic phases over a large temperature range. The first step was fast (k1 = 0.16 min-1) and not very dependent on temperature. The second step was up to two orders of magnitude slower at low temperatures (2 degrees C), but approached the rate of the first step at higher temperatures (40 degrees C). The activation energy for this process was 46 +/- 4 kJ/mol. A third slow process (k3 = 0.9 x 10(-2) min-1 at 40 degrees C) was observed at the higher temperatures. These results suggest that at least two membrane-bound intermediates exist when OmpA folds and inserts into lipid bilayers. We also show that both membrane-bound intermediates can be stabilized in fluid lipid bilayers at low temperatures. These intermediates share many properties with the adsorbed/partially inserted form of OmpA that was previously characterized in gel phase lipid bilayers [Rodionova et al. (1995) Biochemistry 34, 1921-1929]. Temperature jump experiments demonstrate, that the low-temperature intermediates can be rapidly converted to fully inserted native OmpA. On the basis of these and previous results, we present a simple folding model for beta-barrel membrane proteins, in which folding and membrane insertion are coupled processes which involve at least four kinetically distinguishable steps.
Links	PubMed Online version:10.1021/bi961478b
Keywords	Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/metabolism; Dimyristoylphosphatidylcholine/metabolism; Electrophoresis, Polyacrylamide Gel; Escherichia coli/chemistry; Kinetics; Lipid Bilayers/metabolism; Models, Molecular; Molecular Weight; Phosphatidylcholines/metabolism; Protein Conformation; Protein Folding; Protein Structure, Secondary; Spectrometry, Fluorescence; Temperature; Tryptophan
edit table

Significance

You can help EcoliWiki by summarizing why this paper is useful

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.

Annotations

<annotationlinks/>

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

References

See Help:References for how to manage references in EcoliWiki.