2 The effect of phospholipids composition on the folding kinetics of outer membrane protein A into model
2.2 Materials and methods
2.3.3 Efficient OmpA refolding into model membranes containing DOPG and DOPE requires the presence of DOPC
I have shown in section 2.3.1 that the employment of model membranes mimicking the composition of OM (constituted out of PE and PG at a molar ratio of 80 to 20) result in significantly low OmpA folding yields and thus cannot be used efficiently for in vitro refolding studies.
Because I wanted to use for my experiments model membranes containing both phospholipid components, which compose the inner leaflet of the OM, I decided to keep the PG content at 20% and to replace a part of PC with a moderate amount of PE. Thus I have prepared lipid bilayers containing PC, PG and PE at a molar ratio of 50, 20 and 30. I compared the OmpA refolding kinetics in bilayers of PC50PG20PE30 with the kinetics in PC80PG20 bilayers,
The set-up of refolding experiments was described in methods sections 2.2.4 and 2.2.5. The results are displayed in Figure 2.5.
The upper panel displays the SDS-gels obtained after OmpA was refolded into PC50PG20PE30 and PC80PG20 bilayers, respectively. The lower panel contains the densitometric analysis of the SDS-gels from the upper panel. The kinetic parameters of OmpA refolding were calculated as described in section 2.2.6 and are comprised in Table 2. 2.
Figure 2.5 Kinetics of OmpA folding and membrane insertion into model membranes containing (i) PC and PG at a molar ratio of 80/20 and (ii) PC, PG and PE at a molar ratio of 50/20/30. Two separate identical samples of OmpA in 8 M urea were first diluted 12-fold into buffer and then without delay 200-fold molar excess of preformed lipid bilayers containing PC/PG or PC/PG/PE at the molar ratios indicated in the figure, was added to each sample, at 30
°C. Aliquots of each reaction were taken after 2, 4, 8, 16, 30, 60, 120 and 180 minutes and buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE. The gels are displayed in theupper panel. The arrows indicate unfolded (U) and folded (F) forms of OmpA. The lower panel display the densitometric analysis of the fraction of folded OmpA determined from SDS-gels shown in the upper panel.
Table 2. 2 Kinetic parameters for insertion and folding of OmpA derived from the plots of Figure 2.5 (lower panel)
Plots AFa kFb(min-1) kSc (min-1) Yield d(%)
PC80PG20 0.63 ± 0.08 0.4 ± 0.06 0.06 ± 0.02 0.80
PC50PG20PE30 0.63 ± 0.09 0.3 ± 0.05 0.041 ± 0.01 0.77
aRelative contributions of fast phase;bRate constant of fast phase;cRate constant of slow phase;dFolding yield of OmpA.
2.4 Discussions
During the present study, I investigated the effect of PLs similar with those composing the OM on the kinetics of OmpA folding and insertion. Model membranes containing various amounts of PC, PG and PE were employed for this purpose.
Several conclusions can be derived from the results obtained during the present investigation.
First, the model membranes mimicking the composition of the outer membrane and containing PE and PG at a molar ratio of 80 and 20 cannot be successfully employed for my in vitrostudies of OmpA refolding due to their very low refolding efficiency.
The tendency of PE to form nonbilayer structures (such as the inverted hexagonal phase) (van den Brink-van der Laan 2004) may be the factor responsible for the inhibition of OmpA folding into PE80PG20 bilayers (see also Figure 1.4 C from section 1.3.2.1).
PE from biological membranes was shown to be organized only transiently in nonbilayers formations and only in order to allow a larger physiological flexibility for the lipid bilayers (De Kruijff et al. 1985; Luzzati 1997). In contrast, the organization of large amounts of PE into nonbilayer structures cannot be avoidedin vitro.
My results show that only model membranes containing moderate amounts of PE are able to provide the lipid environment necessary for effective in vitroOmpA folding. In addition, the presence of PC is also a requirement for OmpA refolding, due to the tendency of this PL to form bilayers. Thus, OmpA refolding was very efficient into lipid bilayers composed of PC, PG and PE at molar ratios of 50, 20 and 30, respectively.
Other significant conclusions of my study are related with the separate effects of PE and PG on OmpA folding and insertion into model membranes. For this investigation I have used lipid bilayers composed of PC/PE and PC/PG, respectively.
On average, I have noticed a more pronounced decrease of folding yields for PC/PE than for PC/PG membranes. As I have already discussed above, this inhibitory effect manifested by bilayers incorporating PE, is probably due to the general tendency of this phospholipid to form nonbilayer structures, especially when present in large amounts (van den Brink-van der Laan, 2004).
Small amounts of PE appear, on average, to result in faster kinetics and higher folding yields when compared with the effect of large quantities of PE included in the bilayers. This stimulatory effect may be explained by an increase in the curvature stress of membrane induced by small amounts of PE (Tamm et al. 2004) (see also Figure 1.4 C from section 1.3.2.1). The increase of curvature stress means that a larger hydrophobic surface is exposed to the protein during folding. Thus, small amounts of PE are probably able to increase the curvature stress of a predominantly PC bilayer, due to their cone-shaped form.
In comparison with PE, PG has a strong tendency to form bilayers. The presence of PG into PC/PG membranes resulted in changes of the OmpA refolding kinetics due to the negative charge of the polar head group. Thus, small amounts of PG stimulate the OmpA folding kinetics. This effect may be induced by the repulsion between negatively charged PG molecules and the increased hydration shell of this headgroup which, leads to more water in the headgroup region of negatively charged PG compared to PC. Thus, the insertion of OmpA from the aqueous phase into the hydrophobic core of the lipid bilayer is facilitated for membranes containing PG.
In contrast, large amounts of PG resulted in decreased folding yields and slower kinetics probably due to a similar, but significantly stronger electrostatic interaction between the negative charges of amino acids and DOPG. This sort of interation may hinder the OmpA absorption to the lipid bilayer surface.