4 The chaperone effect of SurA on the folding and insertion of outer membrane protein A
4.3.5 Kinetics of tertiary structure formation of OmpA by electrophoresis (KTSE)
4.3.5.1 KTSE method
The KTSE method consists in monitoring the kinetics of tertiary structure formation of OmpA by SDS-PAGE. After purification OmpA is completely unfolded in 8 M urea. The refolding of OmpA is initiated by diluting the urea in buffer at least 12-fold, followed by the addition of preformed lipid bilayers. After the initiation of refolding, samples are taken at different times from the refolding reaction and mixed with an equal volume buffer, pH 6.8. The SDS-buffer contains 0.125 M Tris-HCl, 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol. SDS prevents further OmpA folding (Kleinschmidt and Tamm 1996). SDS-PAGE is performed as described previously but without heat denaturation of the samples (Laemmli 1970). On SDS-PAGE gel the folded OmpA migrates at 30 kDa while the unfolded form of the protein migrates at 35 kDa. The difference in the apparent molecular mass is used to determine the fraction of folded OmpA by densitometry (Kleinschmidt and Tamm 1996). The fraction of folded OmpA resulted from the densitometric analysis is plotted as a function of time. I used the KTSE method to study the chaperone effect of SurA on OmpA insertion and folding.
The next sections (4.3.5.2. to 4.3.5.8) describe the experiments performed during this study, which are based on KTSE method.
4.3.5.2 OmpA refolding in the presence of SurA
The OmpA stock was in 8 M urea (137 µM, pH 7). Samples of 5 µl (24 µg) were taken from the OmpA stock and each was diluted separately into 56 µl HEPES buffer (10 mM, pH 7.0,
with 2 mM EDTA) in the presence and respectively in the absence of a 2-fold molar excess of SurA. Two parallel series each of 5 identical OmpA solutions were prepared. The refolding of OmpA was initiated by adding to each solution a 200-fold molar excess of preformed lipid bilayers (35 µl) containing PC and PG at a molar ratio of 4/1. The lipid bilayers were added to each solution from a series after different intervals of time: 0, 30, 60, 120 and 960 minutes respectively. The final concentration of OmpA was 7.1 µM in a total volume of 96 µl per solution. The OmpA refolding was performed at 37°C. Aliquots of each reaction were taken after 4, 8, 16, 30, 60, 120, 180 and 240 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE.
4.3.5.3 Refolding of aqueous form of OmpA in the presence of SurA
OmpA stock used for this experiment is 1.29 mM in 8 M urea. OmpA of aqueous form was prepared immediately before using it by diluting 20-fold the OmpA stock into HEPES buffer without urea (10 mM, pH 7.0, with 2 mM EDTA). Samples of 8µl (18 µg) were taken from the dilution of aqueous form of OmpA and each was mixed separately with 72 µl HEPES buffer with and without a 4-fold molar excess of SurA. Two parallel series each of 5 identical OmpA solutions were prepared. The refolding of OmpA was initiated by adding to each solution a 200-fold molar excess of preformed lipid bilayers (35 µl) containing PC, PG and PE at a molar ratio of 50/20/30. The lipid bilayers were added to each solution from a series after different intervals of time: 0, 30, 60, 120 and 1440 minutes, respectively. The final concentration of OmpA was 5.35 µM in a total volume of 96 µl per solution. The OmpA refolding was performed at 30°C. Aliquots of each reaction were taken after 4, 8, 16, 30, 60, 90, 120 and 180 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE.
4.3.5.4 OmpA refolding at different SurA / OmpA ratios
The OmpA stock was in 8 M urea (137 µM, pH 7). Separate samples of 5 µl (24 µg) were taken from the OmpA stock and each was diluted into 56 µl HEPES buffer (10 mM, pH 7.0, with 2 mM EDTA) containing SurA at a molar SurA/OmpA ratio of 0, 1, 2 and 4,
Thus, four parallel OmpA solutions were prepared. After 60 minutes a 200-fold molar excess of preformed lipid bilayers (35 µl) containing PC and PG at a molar ratio of 4/1 were added at each solution.
The OmpA was refolded at 37°C. The final solution of OmpA was 7.1 µM in a total volume of 96 µl. Aliquots of each reaction were taken after 4, 8, 16, 30, 60, 90, 120 and 180 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE.
4.3.5.5 OmpA refolding at different temperatures
The OmpA stock was in 8 M urea (137 µM, pH 7). Samples of 5 µl (24 µg) were taken from the OmpA stock and each was diluted separately into 56 µl HEPES buffer (10 mM, pH 7.0, with 2 mM EDTA) in the presence and respectively in the absence of a 2-fold molar excess of SurA. Two parallel series each of 6 identical OmpA solutions were prepared. The dilutions were performed at 5, 15, 25, 30, 37 and 42°C, respectively. The refolding of OmpA was initiated after 60 minutes by adding to each solution a 200-fold molar excess of preformed lipid bilayers (35 µl) containing PC and PG at a molar ratio of 4/1. The final concentration of OmpA was 7.1 µM in a total volume of 96 µl per solution. OmpA from each sample was folded at the temperature of the dilution step. Aliquots of each reaction were taken after 4, 8, 16, 30, 60, 120, 180 and 240 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE.
4.3.5.6 OmpA refolding into lipid bilayers containing different ratios of PC and PG
The OmpA stock was in 8 M urea (137 µM, pH 7). Samples of 5 µl (24 µg) were taken from the OmpA stock and each was diluted separately into 56 µl HEPES buffer (10 mM, pH 7.0, with 2 mM EDTA) in the presence and respectively in the absence of a 2-fold molar excess of SurA. Two parallel series each of 5 identical OmpA solutions were prepared. The refolding of OmpA was initiated after 60 minutes by adding to each solution a 200-fold molar excess of preformed lipid bilayers (35 µl) containing different molar ratios of PC and PG. I used the molar PC / PG ratios of 100/0, 80/20, 50/50, 20/80 and 0/100, respectively. The final concentration of OmpA was 7.1 µM in a total volume of 96 µl per solution. The OmpA was
refolded at 37°C. Aliquots of each reaction were taken after 4, 8, 16, 30, 60, 120, 180 and 240 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE.
4.3.5.7 OmpA refolding into lipid bilayers of various compositions
The OmpA stock was in 8 M urea (137 µM, pH 7). Samples of 5 µl (24 µg) were taken from the OmpA stock and each was diluted separately into 56 µl HEPES buffer (10 mM, pH 7.0, with 2 mM EDTA) in the presence and respectively in the absence of a 2-fold molar excess of SurA. Two parallel series each of 5 identical OmpA solutions were prepared. The refolding of OmpA was initiated after 60 minutes by adding to each solution a 200-fold molar excess of preformed lipid bilayers (35 µl). The composition of the lipid bilayers added to each sample from a series was different. I have used lipid bilayers constituted of PC, PG and PE at the molar ratios of: PC: 100, PC/PG: 80/20, PC/PG/PE: 50/20/30, PC/PE: 70/30 and PE/PG:
80/20. The final concentration of OmpA was 7.1 µM in a total volume of 96 µl per solution.
The OmpA was refolded at 37°C. Aliquots of each reaction were taken after 4, 8, 16, 30, 60, 120, 180 and 240 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE.
4.3.5.8 OmpA refolding in the presence of LPS and SurA
The OmpA stock was in 8 M urea (137 µM, pH 7). Samples of 5 µl (24 µg) were taken from the OmpA stock and each was first diluted 12-fold into buffer either with or without a 2-fold molar excess of SurA. Three solutions were generated, two containing SurA and OmpA and the third one containing only OmpA. 13 µg LPS (10 µl, 342 µM) were then immediately added at one solution with and at one without SurA, at a molar LPS / OmpA ratio of 5/1. An equivalent volume of buffer (10 µl) replaced LPS for the remaining OmpA solution. After LPS addition the solutions are OmpA/SurA/LPS = 1/2/5 (mol/mol/mol), OmpA/SurA = 1/2 (mol/mol) and OmpA/LPS = 1/5 (mol/mol). A 200-fold molar excess of preformed lipid bilayers (26 µl) containing PC and PG at a molar ratio of 4/1 was added immediately to each solution, at 37°C. In a parallel experimental set the lipid bilayers were added after a 60
Aliquots of each reaction were taken after 4, 8, 16, 30, 60, 120, 180 and 240 minutes and SDS-buffer was added to stop further OmpA folding. The samples were then analyzed by SDS-PAGE.
4.3.5.9 Kinetics densitometric analysis and rates of OmpA folding
To determine the kinetic parameters of membrane insertion and folding of OmpA into lipid bilayers the SDS-gels were analyzed by densitometry to obtain the plots of the fractions of folded OmpA as a function of time. The point zero was added at each plot of the fractions of OmpA as a function of time. The point zero represents the unfolded OmpA.
For the present study I assumed that OmpA folding kinetics are accurately described by a two-step mechanism with two pseudo-first order components: a fast phase followed by a slow phase. Thus, I fitted my plots to double-exponential functions. The equation used to fit the plots of the fraction of folded OmpA as a function of time is:
[
PF] (
t)
=FK*[
AF*exp(
-kF*t)
+(
1-AF)
*exp(
kS*t)
- 1]
(Eq.4.1)In Eq.4.1