PD-BamA has no effect on OmpA folding into overall neutral

al., 2009). To explore whether PD-BamA can compensate this inhibitory effect of DOPE, parallel folding experiments were performed with zwitterionic but overall neutral lipid bilayers of the composition DOPC/DOPE (8:2). The lipid concentration was varied to obtain a 200-, 400- and 600-fold molar excess over OmpA in the absence and in the presence of PD-BamA. The fractions of OmpA that were folded at time t after dilution of urea-unfolded OmpA with preformed lipid membranes were analyzed by SDS-PAGE and densitometry and were plotted as a function of time t (Figure 3.2). The analysis (Figure 3.2(A)) indicated that rates and yields of OmpA folding in the absence of PD-BamA (Figure 3.2(B)) were comparable to those obtained in the presence of PD-BamA (Figure 3.2(C)), indicating that PD-BamA has no effect on the folding of OmpA into these lipid bilayers. Interestingly, folding rates and yields increased with an increasing concentration of the DOPC/DOPE membranes. Final folding yields of OmpA were estimated around ~13 % at a lipid/OmpA molar ratio of 200 and increased up to ~32 % at 400- and ~44 % at 600-fold molar excess of lipid (Table 3.2).

M_r (kDa) M_r (kDa) 50

50

50

50

50

50 37

37

37

37

37

37

A

UF

U

U F

F

UF

UF PD-BamA

PD-BamA

PD-BamA UF 4 8 16 30 60 120 180 240

4 8 16 30 60 120 180 240

b a

c

f e d

Time (min)

Fraction of folded OmpA

1.0

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

0.2

0.0

250 200 150 100

250 200 150 100 50 0

B

C

Fraction of folded OmpA

Time (min)

50 0

200 400 600

DOPC/PE (8:2)/OmpA

Figure 3.2 PD-BamA has no effect on the folding of OmpA into neutral lipids. (A) Folding experiments of OmpA into lipid bilayers composed of DOPC/DOPE (8:2) at different lipid concentrations (1.42 mM, 2.84 mM, and 4.26 mM) were performed either in the absence (gels a-c) or in the presence of 14.2 µM PD-BamA (gels d-f), respectively. All folding experiments of urea-denatured OmpA were performed in parallel at 30 °C, and pH 7. Folding was monitored over a time period from 4 to 240 min. The final concentrations of the proteins were 7.1 µM OmpA, and 14.2 µM PD-BamA. The analysis of OmpA folding was done by gel electrophoresis. The gels show the time courses of OmpA folding and the migration of PD-BamA, unfolded OmpA (U) and folded OmpA (F). The fractions of folded OmpA were quantified by densitometry and plotted as a function of time for samples without PD-BamA (B) (gels a to c) and with PD-BamA (C) (gels d to f). The lipid concentrations were 1.42 mM (●), 2.84 mM (◼), and 4.26 mM ( ). Eq. 3.1 was fitted to the experimental kinetics.

The contribution of Af increased in both cases with increasing lipid concentration, whereas the rate constant of the faster process kf remained almost constant. Compared to the observed folding yields obtained with charged lipids, folding yields in DOPC/DOPE (8:2) were reduced, supporting the recent assumption of an inhibiting effect of DOPE. Since PD-BamA was found not to facilitate OmpA folding, it also can be concluded that PD-BamA cannot compensate the inhibitory effect of DOPE in lipid bilayers composed of DOPC/DOPE (8:2).

3.4.2.2 PD-BamA has a strong effect on OmpA folding in the presence of Phos- phatidylglycerol

The kinetics of the folding of OmpA so far demonstrated that PD-BamA facilitated folding and insertion of OmpA into negatively charged ternary lipid mixtures of DOPC/DOPE/DOPG (5:3:2), but not into overall neutral and zwitterionic DOPC/DOPE (8:2) lipids. To examine whether this effect can be attributed to the presence of the negatively charged DOPG, folding experiments with lipids composed of DOPC/DOPG (8:2) were performed at various molar ratios of lipids/OmpA.

Figure 3.3 shows the gels and their densitometric analysis for these OmpA folding experiments, which were performed in the presence of a 200-, 400-, or 600-fold molar excess of SUVs of the composition of DOPC/DOPG (8:2). Analysis of the reactions performed in the presence of PD-BamA (Figure 3.3(C)) showed similar folding rates and yields of folded OmpA (Figure 3.3(C)). The final folding yields of OmpA at all lipid concentrations were estimated around ~82 % in the presence of PD-BamA. In contrast in the absence of PD-BamA, the folding kinetics of OmpA were slower. The final folding yields of OmpA decreased with an increasing lipid concentration from

~47 % to ~20 % at a 200- and at a 600-fold molar excess of DOPC/DOPG (8:2), respectively. The effect of PD-BamA was reflected in the parameters of the folding kinetics obtained from fitting Eq. 3.1 to the experimental data, since Af, kf and ks were all greater in the presence of BamA (Table 3.2). The contribution of the faster folding process, Af, was increased but remained almost constant. This differed remarkably to the observations obtained for the folding of OmpA into bilayers with the ternary lipid composition of DOPC/DOPE/DOPG (5:3:2), where the contribution of the faster folding process increased with increasing lipid concentration. In the absence of PD-BamA, both Af and the rate constant of the faster folding process, kf, decreased with increasing lipid concentration and in all reactions the faster folding process predominated.

M_r (kDa) M_r (kDa) 50

50

50

50

50

50 37

37

37

37

37

37

A

UF

U

U F

F

UF

UF PD-BamA

PD-BamA

PD-BamA UF 4 8 16 30 60 120 180 240

4 8 16 30 60 120 180 240

b a

c

f e d

Time (min)

Fraction of folded OmpA

1.0

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

0.2

0.0

250 200 150 100

250 200 150 100 50 0

B

C

Fraction of folded OmpA

Time (min)

50 0

200 400 600

DOPC/PG (8:2)/OmpA

Figure 3.3 OmpA folding into negatively charged bilayers is strongly facilitated in the presence of PD-BamA. (A) Folding of OmpA (7.14 µM) into lipid bilayers was initiated by a 12-times dilution of urea-denatured OmpA into preformed lipid bilayers composed of DOPC/DOPG (8:2) at different concentrations (1.42 mM, 2.84 mM, and 4.26 mM) either in the absence (gels a-c) or in the presence of 14.2 µM PD-BamA (gels d-f), respectively.

Reactions were performed in parallel at 30 °C and pH 7 and monitored from 4 and 240 min after initiation of folding. Folding of urea denatured OmpA was analyzed by SDS-PAGE. The gels show the time courses of OmpA folding and the migration of PD-BamA, unfolded OmpA (U) and folded OmpA (F). The fractions of folded OmpA in the absence of PD-BamA (B) and in the presence of PD-BamA (C) were analyzed by densitometry. The various lipid concentrations were 1.42 mM (●), 2.84 mM (◼), and 4.26 mM ( ). Eq. 3.1 was fitted to the experimental kinetics.

The results obtained for OmpA folding into bilayers of DOPC/DOPG (8:2) in the absence of PD-BamA are comparable to results obtained with DOPC/DOPE/DOPG (5:3:2) membranes. In conclusion, DOPG seems to be required for PD-BamA to facilitate the kinetics of folding and insertion of OmpA.

3.4.2.3 The presence of PD-BamA supports OmpA folding into membranes composed of DOPE/DOPG (8:2)

Interactions were investigated between PD-BamA and OmpA in a membrane system composed of lipids with native lipid head groups and therefore comparable to the inner leaflet of the outer membrane of E. coli. Experiments on OmpA folding were performed with DOPE/DOPG (8:2) membranes, lacking phosphatidylcholine, which is not naturally occurring in the E. coli OM.

The fractions of OmpA that were folded at time t, obtained by SDS polyacrylamide gels (Figure 3.4, A), were plotted as a function of time t and Eq. 3.1 was fitted to the data (Figure 3.4, B). Experiments with lipid bilayers of DOPE/DOPG (8:2) in the absence and in the presence of PD-BamA were again performed in parallel and the time courses are shown in Figure 3.4. In the presence of PD-BamA, the final folding yields of OmpA were increased by the factor ~3 at 200-, and by the factor ~5 at 400- and 600-fold molar excess of membranes compared to the absence of PD-BamA. In the absence of PD-BamA, OmpA folding was only observed in the presence of a lower lipid concentration at lipid/OmpA molar ratio of 200 and OmpA did not fold into the lipid bilayers at higher lipid concentrations (2.84 mM and 4.26 mM). In contrast, in the presence of PD-BamA the final yields of folded OmpA increased up to a 400-fold molar excess of lipids and were ~46 % (Table 3.2). An increased lipid/OmpA molar ratio of 600 resulted in similar OmpA folding yields and the folding rates were fastest. Neither the fast nor the slow process of the folding of OmpA depended on the lipid concentration. In the absence of PD-BamA, fitting algorithms did not converge when attempting to fit Eq. 3.1 to the data obtained at higher lipid concentrations due to low folding rates and yields. OmpA folding and insertion into DOPE/DOPG membranes were enhanced when PD-BamA was present.

This is quite remarkable, because the fraction of DOPE in the membranes was much increased in comparison to the previous experiments (80 % vs 30 % and 20 %) (Figures 3.2 and 3.4). When PD-BamA was absent, folding yields were strongly diminished in DOPC/DOPG (8:2) in comparison to the ternary lipid mixture (Figure 3.1). The decline of OmpA folding at higher lipid concentrations in the absence of PD-BamA is consistent with previous observations. The general retardation of the kinetics and decline of the folding yields could also be caused by the inhibitory effect of DOPE.

M_r (kDa) M_r (kDa) 50

50

50

50

50

50 37

37

37

37

37

37

A

UF

U

U F

F

UF

UF PD-BamA

PD-BamA

PD-BamA UF 4 8 16 30 60 120 180 240

4 8 16 30 60 120 180 240

b a

c

f e d

Time (min)

Fraction of folded OmpA

1.0

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

0.2

0.0

250 200 150 100

250 200 150 100 50 0

B

C

Fraction of folded OmpA

Time (min)

50 0

200 400 600

DOPE/PG (8:2)/OmpA

Figure 3.4 The inhibiting role of DOPE on the folding of OmpA is reduced by PD-BamA when the negatively charged DOPG is also present in the membrane. (A) Folding experiments of OmpA into lipid bilayers were performed by diluting urea-denatured OmpA 12-times into preformed lipid bilayers composed of DOPE/DOPG (8:2) at different concentrations either in the absence (gels a-c) or in the presence of PD-BamA (gels d-f), respectively. All reactions were performed in parallel at 30 °C and pH 7 between 4 and 240 min. The final concentrations of the proteins were 7.1 µM OmpA, and 14.2 µM PD-BamA. The SDS polyacrylamide gels show the time courses of OmpA folding and the migration of PD-BamA, unfolded OmpA (U) and folded OmpA (F). The fractions of folded OmpA in the absence of PD-BamA (B) and in the presence of PD-BamA (C) were analyzed by densitometry. The various lipid concentrations were 1.42 mM (●), 2.84 mM (◼), and 4.26 mM ( ). Folding kinetics were fitted by Eq. 3.1.

There is little or no effect of PD-BamA on the folding of OmpA into overall neutral bilayers of the zwitterionic lipids DOPC and DOPE. In contrast, in the presence of the negatively charged lipid DOPG, PD-BamA strongly facilitates folding and membrane insertion of OmpA. Consequently, the presence of the negatively charged DOPG in the membrane is a requirement for PD-BamA to facilitate folding of OmpA.

Table 3.2 Kinetic parameters of folding and insertion of OmpA into lipid bilayers with different headgroups^a.

c_Lipid (mM)^c Lipid/

OmpA^d A_f k_f (min^-1) k_s (min^-1) yield (%)^b (A) DOPC/DOPG 8:2

−PD-BamA

1,42 200 0.391 ± 0.016 0.056 ± 0.004 0.0006 ± 0.0002 47 2,84 400 0.171 ± 0.020 0.021 ± 0.003 0.0006 ± 0.0001 28 4,26 600 0.135 ± 0.073 0.023 ± 0.006 0.0004 ± 0.0003 20

+PD-BamA

1,42 200 0.611 ± 0.026 0.088 ± 0.007 0.0042 ± 0.0005 84 2,84 400 0.637 ± 0.035 0.105 ± 0.013 0.0035 ± 0.0008 81 4,26 600 0.679 ± 0.033 0.099 ± 0.011 0.0025 ± 0.0008 82 (B) DOPC/DOPE 8:2

−PD-BamA

1,42 200 - - - 13

2,84 400 0.061 ± 0.009 0.081 ± 0.026 0.0014 ± 0.00006 32 4,26 600 0.125 ± 0.032 0.031 ± 0.010 0.0019 ± 0.00020 44

+PD-BamA

1,42 200 - - - 14

2,84 400 0.120 ± 0.061 0.022 ± 0.013 0.0011 ± 0.0003 33 4,26 600 0.195 ± 0.057 0.022 ± 0.007 0.0016 ± 0.0003 44 (C) DOPE/DOPG 8:2

−PD-BamA

1,42 200 0.052 ± 0.004 0.084 ± 0.017 0.0003 ± 0.00003 11

2,84 400 - - - 1

4,26 600 - - - 1

+PD-BamA

1,42 200 0.169 ± 0.01 0.093 ± 0.018 0.0012 ± 0.0001 36 2,84 400 0.245 ± 0.01 0.144 ± 0.023 0.0014 ± 0.0001 46 4,26 600 0.275 ± 0.02 0.058 ± 0.007 0.0013 ± 0.0002 46

a Urea-unfolded OmpA was folded into SUVs composed of various lipid compositions at various lipid concentrations in the absence and presence of PD-BamA. The data were fitted by Eq. 3.1.

b Yield of folded OmpA after 240 min.

c concentration of the lipid

d Lipid to protein ratio

Im Dokument The periplasmic domain of the barrel assembly machinery protein A (BamA) from Escherichia coli assists folding of outer membrane protein A (Seite 94-101)