3. Dependence of the facilitated folding and membrane insertion of OmpA on the lipid
3.4 Results
3.4.4 Folding studies on OmpA in the presence of BamB and the periplasmic
The POTRA domains of BamA were assumed to serve as scaffold for the interaction with individual BAM subunits and to play a role in the recognition of substrates e.g. unfolded OmpA. BamB is associated to BamA through interactions with POTRA 2 - 5 and was presumed to stabilize the periplasmic domain of BamA (Kim et al., 2007; Chen et al., 2016).
A function arising from interactions of BamB and PD-BamA was investigated on the folding and insertion of OmpA into the lipid membrane. To observe the ability of BamB to assist the folding of OmpA, the experiments were performed using the lipids DLPC, DLPG and DLPE/DLPG (4:1) introducing PD-BamA and a combination of BamB and PD-BamA in the absence and in the presence of NaCl.
The folding of OmpA into noncharged lipids (DLPC) was improved in the presence of either BamB, PD-BamA or BamB/PD-BamA (Fig. 3.15, Table 3.7). The addition of BamB increased the folding yield of OmpA by 19 % while the presence of either BamA or PD-BamA/BamB led to an improvement of 22 %. These results were consistent with the contribution of the faster folding process, Af ~ 0.95 in the presence of BamB and Af ~ 1 in the presence of PD-BamA or of both, BamB and PD-BamA. For comparison, Af ~ 0.71 was calculated from data for the folding of OmpA into pure lipid bilayers. The folding kinetics of OmpA were slower in the presence of NaCl as observed before (see section 3.4.2) but NaCl did not influence the facilitated folding of OmpA into DLPC in the presence of BamB or BamA. The folding yields of OmpA increased from 64 % in the absence of BamB or PD-BamA to 82 % and to 85 % in the presence of BamB and PD-PD-BamA, respectively. In DLPC,
Table 3.6. Rate constants and contribution of the faster folding process to the folding and insertion of OmpA into lipid bilayers of various lipid compositions in the absence and in the presence of NaCl and BamB a.
NaCl Protein Af b kf (min–1) c ks (min–1) d yield (%) e - - 0.397 ± 0.010 0.081 ± 0.004 0.002 ± 0.000 62 - + ProBamB 0.576 ± 0.031 0.101 ± 0.011 0.005 ± 0.001 86 - + BamB 0.573 ± 0.020 0.095 ± 0.007 0.004 ± 0.000 84
+ - - - - 5
+ + ProBamB - - - 15
+ + BamB 0.068 ± 0.040 0.017 ± 0.010 0.000 ± 0.000 12
a Urea-unfolded OmpA (5 μM) was folded into a 200-fold molar excess of DLPE/DLPG (4:1) in the absence and presence of 100 mM NaCl and/or periplasmic ProBamB or cytoplasmic BamB (2-fold excess). Folding kinetics were recorded at 30°C and pH 8.0. Af,kf and ks were obtained by fitting the experimental data from Fig. 3.10 - 3.12 by Eq. 3.1. b Contribution of the faster folding process. c Rate constant of the faster folding process. d Rate constant of the slower folding process. e Final yield of folded OmpA after 240 min.
the facilitated folding and insertion of OmpA in the presence of either BamB or PD-BamA was comparable and independent of the presence of 100 mM NaCl. A combination of both proteins resulted in folding yields and kinetics of OmpA with 97 % folded protein and Af = 0.977 after 240 min. Hence, a combined effect of BamB and PD-BamA on the folding of OmpA was observed in the presence of NaCl.
Fig. 3.15 Effect of BamB and PD-BamA on the folding of OmpA into DLPC in the absence and in the presence of NaCl. Urea-unfolded OmpA (5 μM) was folded into lipid bilayers (1 mM) composed of DLPC in the absence and presence of BamB or PD-BamA (2-fold molar excess) or 100 mM NaCl (30°C, pH 8).
The SDS-polyacrylamid gels (A, C) show the time courses of OmpA folding into DLPC alone (a, e), in the presence of BamB (b, f), in the presence of PD-BamA (c, g) and in the presence of BamB and PD-BamA (d, h) in the absence and presence of NaCl, respectively. Folded OmpA (F) migrates at 30 kDa, unfolded OmpA (U) at 35 kDa, BamB (B) at 42 kDa and PD-BamA (A) at 46 kDa. (B). Upon densitometric analysis the fraction of folded OmpA was determined for each lane and plotted as a function of time (B, D). Also shown are the fits of Eq. 3.2 (experiments without NaCl in the presence of PD-BamA or BamB/PD-BamA) or Eq. 3.1 (remaining experiments) to the experimental data.
Time (min) Time (min)
U U
U
F F
F 35
35
35 kDa
4 8 16 30 60 120 180 240
A
C
35 35
35 kDa
4 8 16 30 60 120 180 240
UF
UF
U F a
b
c
e
f
g d
U F
B
h
35 U
F
D
1.0
0.8
0.6
0.4
0.2
0.0
Fraction of folded OmpA
- + BamB + PD-BamA + BamB + PD-BamA
1.0
0.8
0.6
0.4
0.2
1.00.0
0.8
0.6
0.4
0.2
0.0
Fraction of folded OmpAFraction of folded OmpA
1.0
0.8
0.6
0.4
0.2
0.0
Fraction of folded OmpA
200 150
100 50
Time (min) + BamB + PD-BamA + BamB + PD-BamA + NaCl
40 55
35 40 55
B
A
A B
40 55
40 55
40 B
B A
A
Spontaneous folding of OmpA into negatively charged lipid membranes composed of DLPG was impaired, resulting in 18 % folded protein over time (Fig. 3.16). The presence of either BamB or PD-BamA improved the folding yields of OmpA to 33 % or 25 %, respectively. In the presence of BamB and PD-BamA, 29 % of folded OmpA were obtained. The kinetic parameters were obtained by fitting a single-exponential fit function (Eq. 3.2) to the data points (Table 3.7) and the results from the folding studies of OmpA in the presence of BamB were already described in section 3.4.2. In the presence of PD-BamA or BamB and PD-BamA these fits did not converge and the obtained errors for Af and kf were large, probably because of the slow folding of OmpA.
By screening the overall negative net charge of the DLPG membrane with NaCl, the folding yields of OmpA increased to 86 %. The presence of BamB did not significantly change the folding yields and kinetics of OmpA and a facilitated folding of OmpA was not observable anymore. In contrast, the presence of PD-BamA or BamB and PD-BamA improved the folding yields of OmpA to 95 % or to 98 %, respectively. No significant difference between the folding kinetics of OmpA in the presence of PD-BamA alone or in the presence of BamB and PD-BamA was observed, since Af as well as kf were within error margin about the same.
Fig. 3.16 Effect of BamB and PD-BamA on the folding of OmpA into DLPG in the absence and in the presence of NaCl. Urea-unfolded OmpA (5 μM) was folded into lipid bilayers (1 mM) composed of DLPG in the absence and presence of BamB or PD-BamA (2-fold molar excess) or 100 mM NaCl (30°C, pH 8).
The SDS-polyacrylamid gels (A, C) show the time courses of OmpA folding into DLPG alone (a, e), in the presence of BamB (b, f), in the presence of PD-BamA (c, g) and in the presence of BamB and PD-BamA (d, h) in the absence and presence of NaCl, respectively. Folded OmpA (F) migrates at 30 kDa, unfolded OmpA (U) at 35 kDa, BamB (B) at 42 kDa and PD-BamA (A) at 46 kDa. (B). Upon densitometric analysis the fraction of folded OmpA was determined for each lane and plotted as a function of time (B, D). Also shown are the fits of Eq. 3.1 (experiments with NaCl) or Eq. 3.2 (experiments without NaCl) to the experimental data.
Folding and insertion of OmpA into lipid bilayers composed of DLPE/DLPG (4:1) resulted in folding yields of 39 % folded protein (Fig. 3.17). The folding yields of OmpA increased in the presence of BamB, PD-BamA and BamB/PD-BamA to 69 %, 64 % and 60 %, respectively. The data points of the studies in the presence of BamB or BamB and PD-BamA were fitted by a double-exponential fit function (Eq. 3.1) while for the studies in the presence of PD-BamA a single-exponential fit function was used (Eq. 3.2). In the absence of BamB or PD-BamA, these fits did not converge to the experimental data. Folding of OmpA in the presence of BamB was faster than in the presence of PD-BamA as shown by the kf values
Time (min) Time (min)
U U
U
F F
F 35
35
35 kDa
4 8 16 30 60 120 180 240
A
C
35 35
35 kDa
4 8 16 30 60 120 180 240
UF
UF
U F a
b
c
e
f
g d
UF
B
h
35 U
F
D
40 55
35 40 55
B
A
A B
40 55
40 55
B
B A
A 1.0
0.8
0.6
0.4
0.2
0.0
Fraction of folded OmpA
- + BamB + PD-BamA + BamB + PD-BamA
1.0
0.8
0.6
0.4
0.2
0.01.0
0.8
0.6
0.4
0.2
0.0
Fraction of folded OmpAFraction of folded OmpA
1.0
0.8
0.6
0.4
0.2
0.0
Fraction of folded OmpA
200 150
100 50
Time (min) - + BamB + PD-BamA + BamB + PD-BamA + NaCl
being 0.123 min–1 and kf = 0.004 min–1 in the presence of BamB and PD-BamA, respectively.
kf further increased in the presence of BamB/PD-BamA to 0.172 min–1.
The addition of NaCl drastically decreased the folding yields of OmpA displaying 12 % folded protein in the absence of BamB or PD-BamA. The presence of the individual BAM proteins did not facilitate the folding and insertion of OmpA leading to folding yields of 15 % and 12 % in the presence of BamB and PD-BamA, respectively. The presence of both BAM proteins improved the folding yields of OmpA by 17 % and kf increased from 0.003 min–1 in the absence of the BAM proteins to 0.012 min–1 in the presence of BamB and PD-BamA. All in all, folding and insertion of OmpA into lipid bilayers composed of DLPC, DLPG and DLPE/DLPG (4:1) was facilitated individually in the presence of BamB or PD-BamA.
Fig. 3.17 Effect of BamB and PD-BamA on the folding of OmpA into DLPE/DLPG (4:1) in the absence and in the presence of NaCl. Urea-unfolded OmpA (5 μM) was refolded into lipid bilayers (1 mM) composed of DLPE/DLPG (4:1) in the absence and presence of BamB or PD-BamA (2-fold molar excess) or 100 mM NaCl (30°C, pH 8). The SDS-polyacrylamid gels (A, C) show the time courses of OmpA folding into DLPE/DLPG (4:1) alone (a, e), in the presence of BamB (b, f), in the presence of PD-BamA (c, g) and in the presence of BamB and PD-BamA (d, h) in the absence and presence of NaCl, respectively. Folded OmpA (F) migrates at 30 kDa, unfolded OmpA (U) at 35 kDa, BamB (B) at 42 kDa and PD-BamA (A) at 46 kDa. (B). Upon densitometric analysis the fraction of folded OmpA was determined for each lane and plotted as a function of time (B, D). Also shown are the fits of Eq. 3.1 (experiments with BamB and NaCl or BamB/PD-BamA and NaCl) or Eq. 3.2 (experiments without NaCl or in the presence of PD-BamA and NaCl) to the experimental data.
Time (min) Time (min)
U U
U
F F
F 35
35
35 kDa
4 8 16 30 60 120 180 240
A
C
35 35
35 kDa
4 8 16 30 60 120 180 240
U F U F
UF a
b
c
e
f
g d
UF
B
h
35 U
F
D
40 55
35 40 55
B
A
A B
40 55
40 55
B
B A
A 40
1.0
0.8
0.6
0.4
0.2
0.0
Fraction of folded OmpA
- + BamB + PD-BamA + BamB + PD-BamA
1.0
0.8
0.6
0.4
0.2
0.01.0
0.8
0.6
0.4
0.2
0.0
Fraction of folded OmpAFraction of folded OmpA
1.0
0.8
0.6
0.4
0.2
0.0
Fraction of folded OmpA
200 150
100 50
Time (min)
+ BamB + PD-BamA + BamB + PD-BamA + NaCl
Table 3.7 Rate constants and contribution of the faster folding process to the folding of OmpA into lipid bilayers of various lipid compositions in the absence and in the presence of BamB and PD-BamAa.
Lipid NaCl Protein Af b kf (min–1) c ks (min–1) d yield (%)e DLPC - - 0.709 ± 0.119 0.036 ± 0.009 0.001 ± 0.002 76
- + BamB 0.953 ± 0.081 0.044 ± 0.007 0.000 ± 0.000 95
- + PD-BamA 1.010 ± 0.084 0.032 ± 0.009 - 98
- + BamB/PD-BamA 0.992 ± 0.045 0.042 ± 0.007 - 98 + - 0.543 ± 0.034 0.057 ± 0.007 0.001 ± 0.000 64 + + BamB 0.757 ± 0.041 0.114 ± 0.015 0.001 ± 0.001 82 + + PD-BamA 0.862 ± 0.106 0.061 ± 0.016 0.000 ± 0.000 85 + + BamB/PD-BamA 0.977 ± 0.061 0.058 ± 0.009 0.000 ± 0.000 97
DLPG - - 0.150 ± 0.019 0.026 ± 0.010 - 18
- + BamB 0.317 ± 0.014 0.029 ± 0.004 - 33
- + PD-BamA 0.353 ± 0.128 0.005 ± 0.003 - 25
- + BamB/PD-BamA 1.080 ± 0.667 0.001 ± 0.001 - 29 + - 0.802 ± 0.021 0.190 ± 0.016 0.001 ± 0.001 86 + + BamB 0.795 ± 0.013 0.261 ± 0.015 0.003 ± 0.001 88 + + PD-BamA 0.860 ± 0.015 0.184 ± 0.009 0.006 ± 0.001 95 + + BamB/PD-BamA 0.890 ± 0.016 0.194 ± 0.009 0.007 ± 0.002 98
DLPE/DLPG - - - - - 39
(4:1) - + BamB 0.219 ± 0.026 0.123 ± 0.033 0.004 ± 0.000 69
- + PD-BamA 0.982 ± 0.169 0.004 ± 0.001 - 64
- + BamB/PD-BamA 0.197 ± 0.008 0.172 ± 0.020 0.003 ± 0000 60
+ - 0.242 ± 0.103 0.003 ± 0.002 - 12
+ + BamB - - - 15
+ + PD-BamA 0.184 ± 0.122 0.005 ± 0.005 - 13
+ + BamB/PD-BamA 0.298 ± 0.030 0.012 ± 0.003 - 29
a Urea-unfolded OmpA (5 μM) was folded into a 200-fold molar excess of DLPC, DLPG or DLPE/DLPG (4:1) in the absence and presence of 100 mM NaCl and/or BamB/PD-BamA (2-fold excess). Folding kinetics were performed at 30°C and pH 8.0. Af,kf and ks were obtained by fitting the experimental data from Fig. 3.15 - 3.17 by Eq. 3.1 or 3.2. b Contribution of the faster folding process. c Rate constant of the faster folding process. d Rate constant of the slower folding process. e Final yield of folded OmpA after 240 min.