2 . 8 . S D S - P A G E
For detection of FeoB protein, samples were separated using 10% T solution running gel (10% acrylamide: bis solution (37.5:1), 375mM Tris pH 8.8, 0.1%
SDS, 0.05% APS, 15.3% TEMED) with a 5% T stacking gel (5% acrylamide:
bis solution, 250mM Tris pH 6.8, 0.1% SDS, 0.05% APS, 0.1% TEMED).
Approximately 30µg total protein (5µg for purified samples) was prepared in loading buffer 4x NuPAGE LDS sample buffer, with or without 3% β-mercaptoethanol, which was diluted to a final concentration of 1x with sample.
Sample with loading buffer was incubated at room temperature for 20 minutes followed by loading and running sample in gel at 80-100V in 1x SDS tank buffer (0.3% Trisma base, 1.876% glycine, 0.1% SDS). All gels were run with Bio-Rad Precision Plus Dual Colour standards. Gels were staining using Coomassie stain (10 % acetic acid, 40% methanol, 1% Coomassie Brilliant Blue R-250), destained in destaining solution (7% acetic acid, 5% methanol) and imaged using ImageQuant LAS 4000.
Page 37 of 88 2 . 9 . W e s t e r n b l o t
Protein detection was performed using Invitrogen iBlot Dry Blotting System and the iBlot Gel Transfer Stacks PVDF Mini. SDS-PAGE was run as per 2.6 SDS-PAGE, without staining/destaining. iBlot was prepared following providers instructions with Anode Stack at the bottom, gel placed on the transfer membrane of the Anode stack followed by a pre-soaked Filter paper (soaked in deionized water). Air bubbles were removed by using the blotting roller. Out of the Cathode stack top was the cathode taken and placed on the pre-soaked Filter paper with the electrode side facing up. At this point it was ensured all bubbles were removed by using the roller again. The disposable sponge was placed with the metal contact on the upper right corner of the lid and the lid was shut then. Transfer was achieved through blotting with Program 0. This program runs for 7 minutes in total and uses 20V for 1 minute, 23V for 4 minutes and 25V for the remaining 2 minutes. Following disassembly of apparatus, transfer was checked with Poncheau S solution, which is washed off with water after positive detection of protein on transfer membrane. To block free membrane protein binding sites, membrane was blocked with 25mL fresh TBST (50mM Tris pH 7.4, 150mM NaCl, 0.1% Tween20) containing 5% skim milk powder for 1 hour. Subsequently the membrane was incubated for 1 hour with TBST and 1/1000 anti-his antibodies. Unbound antibody solution was removed through washing of the membrane with TBST for 10 minutes, twice.
Membrane was incubated for 1 hour with 1/5000 anti-mouse antibodies.
Unbound antibody solution was removed by washing with TBST for 10 minutes, three times. For detection of antibody conjugation 2mL each of ECL reagents 1 and 2 was added and left for 5 minutes. Excess reagent was run off the membrane and the membrane wrapped in cling film. Membrane was imaged using ImageQuant LAS 4000 with 1 minute exposure for ISOV and 0.5 to 1 second exposure for pure protein blots.
Page 38 of 88 2 . 1 0 .C r o s s l i n k i n g w i t h d i b r o m o b i m a n e
Purified Protein was reduced by incubation with 5x molar excess of DTT for 20 minutes. 25µg of protein was then diluted in 200µl of assay buffer (10mM K-HEPES, 10% Glycerol, 200mM NaCl, 0.05% C12E8, 10mM MgSO4, 5mM Ascorbic Acid and filled up with MilliQ to 200µl). Half the protein samples and buffer were incubated with 1 mM GTP and 100µM FeSO4 for 20 minutes at RT under slight shaking. Pre-incubated and non-treated samples each were pipetted in dublicates onto a black 96 well plate with clear, flat bottom. The fluorescent probe bBBr was added in 10x molar excess to each well and the plate was immediately read with BioTek Cytation 5 imaging reader using the program Gen5 Image with continuous linear shaking, excitation wavelength of 393nm, emission wavelength of 475nm and the optics in top position. The fluorescence was measured every 10 minutes for a total of 3 hours.
2 . 1 1 .C r o s s l i n k i n g o f p u r i f i e d F e o B
Purified protein was prepared to a final concentration of 20µg in assay buffer (10mM K-HEPES, 200mM NaCl, 0.05% C12E8, 10mM MgSO4, 5mM Ascorbic Acid) a total volume of 30µl. Prior to crosslinking the samples were incubated with either 2mM GTP or 2mM non hydrolysable GTP-gamma-analogue, 100µM FeSO4 and 1mM Protease Inhibitor cocktail for 20 minutes under mild shaking at RT in order to open the pore. Either bifunctional (BMOE) or trifunctional (TMEA) cross linker was added in 5 times molar excess of protein and incubated at RT for 1 hour (bifunctional, BMOE) or 2 hours (trifunctional, TMEA). The crosslinking reaction was stopped by addition of 30µM DTT.
Following the crosslinking 5µg protein sample was mixed with 3µl deionised water and 3µl loading buffer and incubated for 20 minutes at RT. After incubation samples were loaded onto a 4-12% gradient SDS-gel (Invitrogen) and run for 40 minutes at 165V in Invitrogen MOPS running buffer. All gels
Page 39 of 88 were run with Bio-Rad Precision Plus Dual Colour standards. Gels were then either stained using Coomassie stain (10 % acetic acid, 40% methanol, 1%
Coomassie Brilliant Blue R-250), destained in destaining solution (7% acetic acid, 5% methanol) or further processed in a Western Blot (see 2.3) and imaged using ImageQuant LAS 4000 with an exposing time of 0.5-1 second.
2 . 1 2 .C r o s s l i n k i n g i n I S O V
ISOV samples were prepared to a total protein concentration of approximately 100µg in a total volume of 50µl. Prior to crosslinking the samples were incubated with either 2mM GTP or 2mM non hydrolysable GTP-gamma-analogue, 100µM FeSO4 and 1mM protease inhibitor cocktail for 1 hour under mild shaking at RT in order to open the pore. Either bifunctional (BMOE) or trifunctional (TMEA) cross linker was added in 5 times excess of protein and incubated at RT for 1 hour (bifunctional, BMOE) or 2 hours (trifunctional, TMEA). The crosslinking reaction was stopped by addition of 30µM DTT.
Following the crosslinking 30µg total protein was mixed with 3µl deionised water and 3µl 4x NuPAGE LDS sample loading buffer and incubated for 20 minutes at RT. After incubation samples were loaded onto Invitrogen 4-12%
gradient SDS gel and run for 40 minutes at 165V in Invitrogen MOPS running buffer. All gels were run with Bio-Rad Precision Plus Dual Colour standards.
Gels were then further processed in a Western Blot (see 2.3) and imaged using ImageQuant LAS 4000 with an exposure time of 30- 60 seconds.
2 . 1 3 .P u r i f i c a t i o n o f E . c o l i p h o s p h o l i p i d s w i t h A c e t o n e / E t h e r w a s h
All work was carried out under inert gas (N2) and in the dark (aluminium foil covering all used containers). Approximately 100mg E. coli total lipids (Avanti Polar Lipids, Inc.) were weighed and resuspended it in 1 to 2 ml chloroform.
Addition of this lipid-solution in droplets to stirring 10ml ice cold acetone
Page 40 of 88 perfused with N2 and 2µl β-mercaptoethanol. The mixture was stirred for at least 3 hours or overnight at 4°C in the dark and under a N2 atmosphere. The mixture was centrifuged for 15 min at 4000rpm and the supernatant removed.
The pellet was then dried by blowing N2 gas over it, followed by resolubilising it in 10ml diethylether and stirring for 15 min at RT. The solution was centrifuged for 20 minutes at 3000rpm and the pellet was discarded. The remaining supernatant was evaporated to dryness in a Büchi rotary evaporator R at RT. The residue was dissolved in 2-3ml chloroform and transferred to pre-weighed glass vessels. The lipids were dried as a thin film under N2 gas. The glass tube was then weighed again and the amount of E. coli phospholipids was calculated. Egg yolk phosphatidylcholine was added in a 1:3 ratio (EYPC : ECPL) and mixture was dissolved in 1ml chloroform and aliquoted into roughly 30mg phospholipids per glass tube. The solution was evaporated to dryness again while rotating the glass tube in order to give a thin layer of phospholipids on surface. Phospholipids were stored under N2 atmosphere and in the dark at –20°C.
2 . 1 4 .P r e p a r a t i o n o f p r o t e o l i p o s o m e s
Lipids were thawn on ice and the film rehydrated to a concentration of 20mg of phospholipids per ml in 50mM KPi pH=7.0 and addition of 5µM Phengreen SK, 200µM FeSO4 and 5mM ascorbic acid to the mixture. The solution was alternately vortexed and rested to gain a homogenous suspension and snap frozen in dry ice/EtOH. Liposomes were then slowly and undisturbed thawn on ice again. Subsequently the liposomes were extruded 11 times through a 400nm polycarbonate filter to form unilamellar liposomes of homogenous size using an Avanti Mini Extruder. The extruded liposomes were diluted to a concentration of 4mg/ml in 50mM KPi pH=7.0. For destabilising the liposomes the detergent TritonX-100 was used. Small aliquots (2.5µl) of a 10% stock were added and absorbance changes were followed at 540nm. Prior to
Page 41 of 88 absorbance measurement after each addition of TX100 an incubation of 30 seconds while stirring was carried out. As soon as a level of saturation (Rsat) was reached another two aliquots were added to make sure the destabilisation is guaranteed, but the liposomes do not get into a critical state of decomposition. At this step purified protein was added to the detergent-doped liposomes in a ratio of 50:1 (Lipid : protein, m|m) and incubated for 30 minutes under mild stirring at RT. Bio-Beads were washed three times with methanol, one time with ethanol and five times with deionised water. Washed bio-beads (80mg per ml liposome-solution) was added and incubated for 2h under mild stirring while protected from light. Bio-beads were allowed to settle down and the solution transferred to a fresh glass tube. Again washed bio-beads (80mg/ml) were added and incubated for 2h under mild stirring, this time at 4°C while protected from light. The last step was repeated and solution incubated over night. After removal of bio-beads the proteo-liposomes were harvested by centrifugation for 30 minutes at 50,000rpm and 4°C. The supernatant was discarded and the pellet resuspended in 100µl 50mM KPi pH=7.0 to give a protein concentration of 1mg/ml, assuming 100%
reconstitution efficacy. Liposomes were used immediately after preparation.
2 . 1 5 .L i p o s o m e A s s a y
For the liposome assay 20µl liposomes were added to 1.980 ml assay buffer (50mM KPi pH=7.0, 10mM MgSO4, 5mM Phosphoenolpyruvate (PEP), 0.5µg Proteinkinase) while stirring. Initially the maximum fluorescence was measured by transferring sample to 2mL glass cuvette, and fluorescence measured with a PerkinElmer LS 55B fluorimeter using program FLWinLab:
excitation wavelength 356nm (10nm slit width), emission 438nm (3nm slit width). Fluorescence was measured for 30 seconds prior to addition of 2mM GTP, after which the fluorescence was measured for the remaining time of 30
Page 42 of 88 minutes. In a separate run with the same setup a following addition of 0.5%
SDS after 15 minutes was performed in order to disrupt the liposomes.
2 . 1 6 .G T P - / A T P - b i n d i n g
Purified protein was prepared in assay buffer (10mM MgSO4, 200mM NaCl, 10mM K- HEPES pH 7.0) to a final concentration of 25µg/ml to a final volume of 2ml. A 50µM stock of fluorescent mantGTP and mantATP was prepared.
Fluorescence measured with a PerkinElmer LS 55B fluorimeter using program FLWinLab: excitation wavelength 500nm (slit 10nm width), emission 534nm (3nm slit width). Fluorescence was measured for 30seconds prior to addition of 0.125µM GTP or ATP respectively every 30 seconds for the remaining time of 20 minutes. Additional to the samples the same setup was used for a negative control (assay buffer only) to test for nonspecific binding. These results were used for normalisation of the sample results and showing the specific binding of the samples to either mantGTP or mantATP.
Page 43 of 88
3 . R e s u l t s
3 . 1 . I s o l a t i o n o f F e o B e x p r e s s i n g p l a s m i d s
For optimal protein expression FeoB was always prepared from freshly transformed DH5α E. coli cells. For transformation pure supercoiled FeoB expressing plasmids were used. In order to test the predicted model, four different plasmids, expressing WT FeoB and three FeoB mutants were prepared (Seyedmohammad et al., 2014). To verify the presence and the yield of plasmids, agarose gel electrophoresis was performed. The results can be seen in Figure 7.
Figure 7: Purification of FeoB expressing Plasmids. Plasmids were purified from DH5α cells using Purelink quick plasmid miniprep kit. The presence of plasmid was confirmed by 1% Agarose gel electrophoresis (2µl per lane). The arrow marks FeoB expressing plasmids.
Page 44 of 88 3 . 2 . P r e p a r a t i o n o f I n s i d e O u t V e s i c l e s
For overexpression of the membrane protein FeoB, the E. coli strain C41(DE3) was used. As membrane proteins show potential toxicity to the expressing cells (Gubellini et al., 2011), this strain is specifically modified for optimal expression of membrane proteins with minimal toxicity. After transformation with FeoB expressing plasmids, the bacteria were used to prepare Inside Out Vesicles (ISOV). The expression of FeoB was verified with a 10% SDS-PAGE with a supposed band at around 83 kDa. As can be seen in Figure 8, the FeoB bands appear at a size of approximately 75kDa. This aberrant effect is common for membrane proteins and observed in a variety of them.
Incomplete unfolding of the protein, different levels of SDS-binding, oligomerization or different post-translational modifications can affect the behaviour of membrane proteins on a SDS gel (Rath et al., 2009).
Previous studies showed that the highest levels of FeoB can be obtained when expressed at 18°C (Seyedmohammad et al., 2014). High levels of expression were obtained for wild type, D123N and C429S FeoB. However, C675S FeoB showed a significant lower expression level compared to the other samples.
Page 45 of 88
Figure 8: Expression of FeoB in C41 (DE3) cells. ISOV were prepared from E. coli C41 cells harbouring the non-expressing construct plasmid for negative control and the FeoB expressing plasmids for WT and mutants of FeoB. Proteins (30µg total protein per lane) were separated on SDS-PAGE (10%). Protein bands were visualised by staining with Coomassie-Blue. The arrow indicates the expression of FeoB in all lanes but the negative control (Non-expressing control).
FeoB
Page 46 of 88 3 . 3 . P u r i f i c a t i o n o f F e o B
FeoB was purified from the ISOV. Membrane proteins are insoluble in aqueous solutions, so it was necessary to solubilise the protein in presence of a detergent. DDM was the detergent of preference as it shows a high level of FeoB stability and does not interfere with its activity when bound. During the purification process DDM was exchanged with the detergent C12E8. This detergent shows better results in stability for long-term storage of the protein (Seyedmohammad et al., 2014). Several steps during purification were checked for success with a 10% SDS-PAGE. The results for WT and mutant FeoB can be seen in Figure 9- Figure 12. The final purified samples of WT, C429S and D123N FeoB showed a concentration of > 5mg/ml FeoB protein per litre of culture and minimal contamination proteins could be obtained. As the third mutant C675S FeoB already showed low ISOV expression levels, a higher amount of ISOV were used to gain same levels of purified proteins. In spite of this adjustment the purified protein levels were much lower compared to the other samples.
Page 47 of 88
Figure 9: Purification of WT FeoB. Purification of WT FeoB was done with affinity chromatography. To check for successful purification samples of all fractions were separated on SDS-PAGE (10%, 30µg of total protein in each lane, except 5µg for pure protein were used). Protein bands were visualised by staining with Coomassie-Blue.
Arrow marks WT FeoB band, showing a high yield of pure WT FeoB protein.
FeoB
Page 48 of 88
Figure 10: Purification of C429S FeoB. Purification of C429S FeoB was done with affinity chromatography. To check for successful purification samples of all fractions were separated on SDS-PAGE (10%, 30µg of total protein in each lane, except 5µg for pure protein were used). Protein bands were visualised by staining with Coomassie-Blue.
Arrow marks C429S FeoB band, showing a high yield of pure C429S FeoB protein.
Figure 11: Purification of
affinity chromatography. To check for successful purification sample were separated on
SDS-pure protein were used). Protein bands were visualised by staining with Coomassie Arrow marks C675S FeoB band, showing a low yield of pure C675S Fe
Purification of C675S FeoB. Purification of C675S FeoB was done with affinity chromatography. To check for successful purification samples of all fractions -PAGE (10%, 30µg of total protein in each lane, except 5µg for pure protein were used). Protein bands were visualised by staining with Coomassie Arrow marks C675S FeoB band, showing a low yield of pure C675S FeoB protein.
Page 49 of 88
Purification of C675S FeoB was done with s of all fractions PAGE (10%, 30µg of total protein in each lane, except 5µg for pure protein were used). Protein bands were visualised by staining with Coomassie-Blue.
oB protein.
Page 50 of 88
Figure 12: Purification of D123N FeoB. Purification of D123N FeoB was done with affinity chromatography. To check for successful purification samples of all fractions were separated on SDS-PAGE (10%, 30µg of total protein in each lane, except 5µg for pure protein were used). Protein bands were visualised by staining with Coomassie-Blue.
Arrow marks D123N FeoB band, showing a high yield of pure D123N FeoB protein.
Page 51 of 88 3 . 4 . G T P - / A T P - b i n d i n g
As previously described, most transporters are ATP powered. FeoB of P. aeruginosa however is thought to bind and hydrolyse GTP instead (Guilfoyle et al., 2009; Hantke, 2003). For conformation WT and mutant FeoB, respectively were incubated with increasing amounts of fluorescent mantGTP and mantATP analogues. These analogues are modified ATP and GTP bound to a fluorescent dye. The binding quenches the fluorescent signal, but as soon as mantATP/-GTP are bound and hydrolysed, the fluorescent dye gets separated.
Thus, the fluorescent signal is not quenched anymore and can be measured.
The changes in fluorescence and therefore binding and hydrolysis of the probes were followed for 20 minutes. To take nonspecific binding into consideration, a negative control consisting of buffer only, was tested under same conditions. Protein samples were normalised to the negative control to show specific binding levels. As shown in Figure 13 and Figure 14, WT and mutant FeoB show almost identical levels in binding of GTP, while no binding could be observed for ATP.
Page 52 of 88
Figure 13: GTP binding of WT and mutant FeoB. The activity of WT and mutant FeoB (25µg each) was measured using the fluorescent GTP-analogue mantGTP. GTP binding was measured as a fluorescent increase at 534nm upon incremental increase in [mantGDP]. The GTP binding activity is almost identical for WT and mutant FeoB.
0 50 100 150 200 250
Fluorescence [RU]
mantGTP [µM]