Crosslinking in pure protein

As shown in Figure 24 the monomeric WT FeoB bands at a size of about 70kDa, indicated with a red square, show a massive decrease in intensity. This decrease was not shown for the C429S FeoB mutant, marked in a blue square.

In contrast to expected results, there were no clearly visible bands for dimers and trimers at the expected sizes.

Page 62 of 88

Figure 24: Crosslinking of WT and C429S FeoB with bi- and trifunctional crosslinker. WT and C429Amutant FeoB were incubated with the bifunctional crosslinker BMOE and and trifunctional crosslinker TMEA in order to crosslink the pore-lining cysteines in an open state of the pore. All samples were separated on a 4%-10%

gradient SDS-PAGE (5µl sample per lane). The protein-bands were visualized with by staining with Comassie Blue. The reduction in the intensity of the WT FeoB bands is marked with a red square. The steady intensity of C429S FeoB bands is marked with a blue square.

kDa

Page 63 of 88 3.5.6. Crosslinking in ISOV

ISOV were used in order to test this crosslinking in a state closer to the original physiological composition. In this composition, the protein is integrated in a membrane which is more in line with the natural environment of the protein. Beside the mentioned co/-factors the WT FeoB ISOV was treated with a non-hydrolysable GTP-analogue in order to see if GTP binding alone is enough for opening the pore.

As can be seen in Figure 25 clearly visible bands indicate the presence of crosslinked dimers and trimers, marked in a red square. This bands were only seen in WT FeoB ISOV treated with GTP. WT FeoB ISOV treated with the GTP-analogue and C429S FeoB ISOV did not show similar bands. However, the bands for WT FeoB ISOV were not at the expected size of 160 and 240 kDa, instead they appear at a much larger size.

Page 64 of 88

Figure 25: Western Blot of WT and C429S FeoB ISOV. The samples were incubated with different combination of substances as shown. Untreated samples were used as negative control. GTP-γ-analogue is a non hydrolysable analogue. It was used to check whether the binding or hydrolysis of GTP leads to an opening of the Pore. Both bi- and trifunctional crosslinkers were used, respectively. The building of di- and trimers was observed in samples treated with Fe and genuine GTP only, marked with the red square.

No other sample showed comparable binding of the crosslinkers.

In order to cross-check the effect on the pore of WT FeoB when binding the GTP-γ-analogue, a similar experiment was carried out with the D123N FeoB mutant. This mutant is able to bind GTP, but is not able to hydrolyse it. WT and mutant FeoB samples were incubated with GTP and iron prior to crosslinking with the bi- and trifunctional crosslinkers. As can be seen in Figure 26 no crosslinking was observed. The intensity of the protein bands neither increases nor are bands of dimers and trimers visible.

Page 65 of 88

Figure 26: Western Blot of D123N FeoB ISOV. This mutant is able to bind GTP, however cannot hydrolyse it. The samples were incubated with Fe and GTP prior binding of the bifunctional crosslinker BMOE and the trifunctional crosslinker TMEA in order to open the pore. No crosslinking was shown in D123N FeoB.

3 . 6 . I r o n t r a n s p o r t

FeoB is predicted to be the active permease of the Feo system, responsible for Fe transport. However, FeoB mediated Fe transport has never been measured directly.

To show FeoB driven iron transport, liposomes carrying iron bound to a fluorescent probe were used. The fluorescent probe phengreen SK binds to metal ions (e.g. iron, copper, nickel), it is not membrane permeable and the

Page 66 of 88 fluorescent signal is quenched when bound. During reconstitution purified FeoB was incorporated into the membrane of the liposomes in an inside-out manner. This orientation is maintained as the cytosolic part of the protein is hydrophile, whereas the periplasmatic and the transmembrane parts are hydrophobe. These preferences lead to the incorporation in the inside out orientation, as the proteoliposomes build up micelles with a polar outside and a nonpolar inside. The inside-out orientation of FeoB is necessary as iron needs to be transported from inside the liposomes to the outside. For easier understanding a scheme is shown in Figure 27.

Figure 27: Scheme of mode of action for the iron transport model. WT FeoB was incorporated in the membrane of phospho-liposomes in an inside-out manner in order to transport Fe²⁺ from the inside of the liposomes to the outside. Within the phospho-liposome Feo²⁺ is bound to the fluorescent dye phengreen SK. If WT FeoB acts as predicted, Fe gets separated from the fluorescent dye and transported to the outside.

Without the quenching by binding, the dye emits a measurable fluorescent signal.

Proteoliposome Proteoliposome

Page 67 of 88 3.6.1. Preparation of proteoliposomes

The presence of FeoB in the proteoliposomes was confirmed with a 10% SDS-PAGE. As can be seen in Figure 28, WT and C429S FeoB were present, marked by a red arrow. The negative control does not contain any protein.

Figure 28: Presence of WT and C429S FeoB in the proteoliposomes. To check for successful incorporation of FeoB into the membrane of the liposomes, WT FeoB, C429S FeoB and empty liposome samples were separated on SDS-PAGE (10%, maximal loading volume of 8µl sample in each lane). Protein bands were visualised by staining with Coomassie-Blue. Arrow marks FeoB band, showing the presence of FeoB protein in the liposomes. Liposome residues marked with a dashed square.

FeoB

Page 68 of 88 3.6.2. Liposome-assay

Inside the liposome iron is bound to a fluorescent probe, phengreen SK, which is not membrane permeable. The fluorescent signal of the probe is quenched as long as it is bound to iron. Following iron transport the probe is not longer bound and the quenching effect is removed, leading to a fluorescence signal.

This assay was used to show iron transport via FeoB. GTP was added to open the pore and enable iron transport. As mentioned earlier FeoB shows a very slow GTP hydrolysis. To prevent the complete consumption of GTP before the pore is opened, a pyruvatekinase and phosphoenolpyruvate was added to the buffer to keep GTP levels constant during the experiment.

Contrary to what was expected, there was no increase in fluorescence measured. After addition of GTP a decrease in fluorescence to almost zero is observed. Results are shown in Figure 29.

Figure 29: Liposome assay. In order to show transport of iron by FeoB the assumed physiological conditions were simulated by addition of 2mM GTP to the samples. The expected result was an increase of fluorescence as iron should be transported out of the liposomes and the remaining unbound fluorescent dye phengreen SK emitting a fluorescent signal. In contrast a further decrease of fluorescence measure was observed.

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Fluorescence [RU]