2 The effect of phospholipids composition on the folding kinetics of outer membrane protein A into model
2.2 Materials and methods
2.3.2 Folding and insertion of OmpA depend on the amount of PG and PE included into model membranes
In the previous section 2.3.1, was shown that model membranes containing exclusively PE and PG cannot be efficiently employed to study the effect of these phospholipids on OmpA refolding. Therefore, binary lipid mixtures of PC/PE and PC/PG were used to investigate the effect of PE and PG on the kinetics of OmpA folding and insertion.
The experiments performed for this purpose had a common set-up: first separate samples of OmpA in 8 M urea were 12-fold diluted in buffer and then the lipid bilayers containing PC/PE or PC/PG respectively, were immediately added at 30°C. I have used lipid bilayers constituted of PC and PE at the molar ratios of: PC100, PC/PE: 90/10, PC/PE: 70/30, PC/PE: 60/40. For the bilayers containing PC and PG the molar ratios were: PC: 100, PC/PG: 80/20, PC/PG:
70/30, PC/PG: 50/50, PC/PG: 30/70.
The appereance of the folded form of OmpA was monitored for 180 minutes, as described in detail in the method sections: 2.2.4 and 2.2.5. The results of OmpA refolding experiments are displayed in Figure 2.3. The upper panel of Figure 2.3 displays the SDS-PAGE gels. The lower panels of Figure 2.3 (A and B) represent the densitometric analysis of SDS-gels from upper panel.
At least two plots obtained from separate experiments performed in identical conditions and different days were averaged in order to generate the graphs from panels A and B, Figure 2.3.
In Figure 2.4 the OmpA folding yields obtained after 180 minutes from OmpA refolding initiation were plotted as a function of the PE and respectively PG amount present in bilayers.
The OmpA folding yields at 180 minutes were obtained from the densitometric analysis of refolding kinetics (Figure 2.3, lower panels, A and B).
The kinetic parameters of OmpA refolding (Table 2.1) were derived from the plots of Figure 2.3 (lower panels A and B) and were calculated as described in the section 2.2.6.
Overall, the data from Figure 2.3, 2.4 and Table 2.1 show that the progressive increase of PE and PG amounts result, in average, in reduced OmpA folding yields and slower refolding kinetics.
In the case of PC/PE bilayers, the decrease of OmpA folding yields after 180 minutes from refolding initiation is more pronounced than for PC/PG bilayers. This difference is notably higher for amounts of PE and PG above 30 % (Figure 2.4 and Table 2.1). Thus, for a 40 % PE the folding yield is 0.46, while a very similar yield 0.47 is obtained for a significantly higher amount of PG: 70 % (Figure 2.4 and Table 2.1).
Figure 2.3 Kinetics of OmpA folding and insertion into model mebranes containing binary mixtures of PC/PE and PC/PG. 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/PE or PC/PG 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 SDS-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.Lower panelsAandBdisplay the densitometric analysis of the fraction of folded OmpA determined from SDS-gels shown in the upper panel.
Table 2. 1 Kinetic parameters for folding and insertion of OmpA derived from the plots of Figure 2.3 (Panels A and B)
Panel %PL AFa kFb(min-1) kSc (min-1) Yieldd(%)
A 0 0.31 ± 0.05 0.25 ± 0.07 0.02 ± 0.004 0.86
10 0.28 ± 0.04 0.33 ± 0.12 0.01 ± 0.004 0.76
20 0.61 ± 0.08 0.18 ± 0.04 0.02 ± 0.010 0.81
30 0.23 ± 0.01 0.84 ± 0.20 0.02 ± 0.001 0.67
40 0.19 ± 0.02 0.82 ± 0.30 0.01 ± 0.002 0.46
B 0 0.31 ± 0.05 0.25 ± 0.07 0.02 ± 0.004 0.86
10 0.15 ± 0.06 0.12 ± 0.06 0.02 ± 0.002 0.85
20 0.61 ± 0.07 0.40 ± 0.06 0.06 ± 0.010 0.80
30 0.10 ± 0.01 0.33 ± 0.12 0.01 ± 0.001 0.82
50 0.03 ± 0.01 0.14 ± 0.001 0.01 ± 0.001 0.70
70 0.23 ± 0.02 0.78 ± 0.33 0.02 ± 0.002 0.47
aRelative contributions of fast phase;bRate constant of fast phase;cRate constant of slow phase;dFolding yield of OmpA.
Interestingly, in the case of 20 % PG bilayers, the kinetics of OmpA refolding appear to be in average stimulated.
Figure 2.4 Yields of folded OmpA depend on the amount of PE and PG present into the model membranes employed for refolding. The OmpA folding yields at 180 minutes were obtained from the densitometric analysis of refolding kinetics (Figure 2.3, lower panels, A and B).
The yields of refolded OmpA were plotted as a function of the PE (blue circles) and PG (black circles) ratios diplayed in Figure 2.3, lower panels, A and B.
The kinetics parameters of refolding (Table 2.1) show that, in average the contribution of the fast phase to the OmpA folding process is very similar for both PE and PG bilayers (Table 2.1).
Averages of the rate constants characterizing the fast process are also similar but the refolding into PE bilayers resulted in slightly higher rate constants in comparison with the refolding into PG membranes (Table 2.1).
Notably, the most efficient folding in terms of dominance of the fast phase of refolding and rate constant was obtained with PC80PG20 bilayers (Table 2.1). In contrast, the experiments employing bilayers with high amounts of PE or PG (above 40 molar percent of lipid) yielded in average the highest rate constants and the lowest folding yields (Table 2.1).
All these results show that OmpA folding and insertionin vitro depends on the amount of PE and PG incorporated into model membranes.
In this respect, best folding results in average were obtained with PC80PG20 bilayers. The presence of a moderate amount of PG, which is negatively charged may stimulate the OmpA refolding into model membrane. The presence of the same amount of PE (20 %) resulted into kinetics comparable with those obtained with PC80PG20 bilayers (Figure 2.3 and table 2.1).
In comparison with PE bilayers, the PG membranes appear on average, to result in higher folding yields and slightly lower rates of refolding.