FOLDING INTERMEDIATES OF A BETA-BARREL MEMBRANE-PROTEIN - KINETIC EVIDENCE FOR A MULTISTEP MEMBRANE INSERTION MECHANISM

The mechanism of folding and membrane insertion of integral membrane p roteins, including helix bundle and beta-barrel proteins is not well u nderstood. A key question is whether folding and insertion are coupled or separable processes. We have used the beta-barrel outer membrane p rotein A (OmpA) of Escherichia coli as a model to study the kinetics o f folding and insertion into dioleoylphosphatidylcholine (DOPC) bilaye rs as a function of temperature by gel electrophoresis, protease diges tion, and fluorescence spectroscopy, OmpA was unfolded in 8 M urea sol ution (without detergent), and refolding and membrane insertion was in itiated by rapid dilution of the urea concentration in the presence of phospholipid vesicles. In addition to the kinetically unresolved hydr ophobic collapse in water, the time course of refolding of OmpA into D OPC bilayers exhibited three kinetic phases over a large temperature r ange. The first step was fast (k(1) = 0.16 min(-1)) and not very depen dent 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 e nergy for this process was 46 +/- 4 kJ/mol. A third slow process (k(3) = 0.9 x 10(-2) min(-1) at 40 degrees C) was observed at the higher te mperatures. These results suggest that at least two membrane-bound int ermediates exist when OmpA folds and inserts into lipid bilayers. We a lso show that both membrane-bound intermediates can be stabilized in f luid lipid bilayers at low temperatures, These intermediates share man y properties with the adsorbed/partially inserted form of OmpA that wa s previously characterized in gel phase lipid bilayers [Rodionova et a l. (1995) Biochemistry 34, 1921-1929]. Temperature jump experiments de monstrate, that the low-temperature intermediates can be rapidly conve rted to fully inserted native OmpA. On the basis of these and previous results, we present a simple folding model for beta-barrel membrane p roteins, in which folding and membrane insertion are coupled processes which involve at least four kinetically distinguishable steps.