5 Results and Discussion
5.2 The role of TtOmp85 in TtoA folding
5.2.5 Immobilization of TtOmp85 via short peptides
A further step in the investigation of TtOmp85/TtoA interaction was the immobiliza-tion of TtOmp85to the IRE surface via short C-terminal TtoA peptides that could interact with the TtOmp85 barrel via β-augmentation but not form an own β-barrel.
The goal of this experiment was to create a stable hybrid barrel between TtOmp85 and TtoA and thus provide evidence for our proposed mechanism of β-augmentation.
We used the one-stranded peptide b8 (Figure 4.12, upper sequence) and the modied two-stranded peptide b7/8∗ (Figure 4.12, lower sequence). Spectra of b8 and b7/8 im-mobilization and TtOmp85 binding were not reproducible. Solubility of b8 and b7/8 was low. An attempt to increase b7/8 solubility by exchange of hydrophobic for hy-drophilic amino acids did not increase solubility signicantly. Figure 5.32, Figure 5.33,
5. RESULTS AND DISCUSSION 5.2. ROLE OF TTOMP85 IN TTOA FOLDING
Figure 5.34 and Figure 5.35 show dierent attempts of peptide immobilization. Fig-ure 5.32 shows a spectrum of b8 after incubation with activated silane-NTA linker and rinsing. Peptide is present on the crystal after rinsing with an intense band band at 1623 cm−1, but it is likely due to precipitation because it was not possible to solubilize it completely in FC-12 buer. Incubation with TtOmp85 and rinsing did not result in major changes of the spectrum except for an increase of a shoulder in the mid-amide I region. It is unlikely that TtOmp85 only this minor eect on the spectrum when it is immobilized to b8. In order to increase b8 solubility 10% FC-12 were used (Figure 5.33). However, neither peptide nor TtOmp85 could be immobilized, as the spectra with undened bands and minima show. This might be due to the high amount of detergent on the crystal, which could hinder binding of the peptide and the protein.
It also seems as if components were removed from the crystal by repeated rinsing.
When peptide b7/b8* is solubilized in 0.5%FC-12 it seems that either immobilization or precipitation take place during incubation with activated silane-NTA linker due to bands at 1627 cm−1 and 1647 cm−1. Since solubilization was dicult precipitation is likely. The incubation with TtOmp85 does not result in major spectral dierences, thus leading to the conclusion that TtOmp85 binding to the peptide did not occur.
Figure 5.35 shows spectra that were recorded after incubation of b7/8* with linker and rinsing. The red spectrum is of TtOmp85 during incubation on the crystal. The blue spectrum is after rinsing and shows that TtOmp85 is removed from the crystal and thus not immobilized by interaction with b7/8*. Estrada et al.[100] showed that short peptide sequences were able to form stable hybrid barrels with TtOmp85. The results presented in this section neither conrm nor refute this. Even when peptide is present on the linker, interaction with TtOmp85 is likely not possible becuase, instead of binding to the activated silane-NTA linker, b8 and b7/8*, respectively, precipitated.
In higher detergent concentrations it was possible to dissolve the peptide but binding to the linker was unsuccessful.
5. RESULTS AND DISCUSSION 5.2. ROLE OF TTOMP85 IN TTOA FOLDING
Figure 5.32: 0.3 mg/mL of peptide b8 were dissolved in Tris buer with 0.1 % FC-12.
Spectra show peptide after incubation with activated silane-NTA linker after rinsing (black) and TtOmp85 after incubation with b8 after rinsing (red).
Figure 5.33: 0.3 mg/mL of peptide b8 were dissolved in Tris buer with 10%FC-12. Spectra show peptide after incubation with activated silane-NTA linker after rinsing (black) and TtOmp85 after incubation with b8 after rinsing (red).
5. RESULTS AND DISCUSSION 5.2. ROLE OF TTOMP85 IN TTOA FOLDING
Figure 5.34: 0.5 mg/mL of peptide b7/8* were dissolved in Tris buer with 0.5% FC-12.
Spectra show peptide after incubation with activated silane-NTA linker after rinsing (black) and TtOmp85 after incubation with b7/8* after rinsing (red).
Figure 5.35: 0.5 mg/mL of peptide b7/8* were dissolved in Tris buer with 5%SDS. Spectra show TtOmp85 during incubation with immobilized or precipitated (red) and after rinsing (blue).
6 Conclusion
Understanding the folding mechanisms and pathways of proteins is a highly investi-gated topic, especially in regard to protein misfolding diseases. Therefore, the doctoral thesis focuses on the investigation of the stability and folding of two outer membrane proteins, TtOmp85 and TtoA. Experiments showed that the melting point of TtoA in absence of denaturants is > 100 ◦C. Thus, extensive studies on TtoA stability were performed, as it was necessary to have access to the unfolded protein for folding ex-periments. These included the application of heat in combination with denaturants on native TtoA. In presence of SDS and urea partial unfolding could be achieved by application of heat but the protein adopted a compact structure when recooled to room temperature. 2-Mercaptoethanol led to complete unfolding of TtoA at 100 ◦C, indi-cating the importance of the extracelluar disulde bridge for TtoA stability. It was not possible, however, to keep TtoA unfolded at room temperature under these condi-tions. Mutation studies with cysteine mutants were inconclusive because the mutants and the control wildtype were expressed as inclusion bodies and seemed to adopt a non-native structure upon folding. Another approach to solve the problem of working with unfolded TtoA was to purify TtoA from inclusion bodies. It was kept in high amounts of SDS and remained mostly unfolded for a limited time and to a limited de-gree at room temperature. However, even under these conditions TtoA always showed signicant amounts of residual structure. In order to allow controlled folding of TtoA in presence of TtOmp85, an experiment was established to spectroscopically observe structural changes in unfolded TtoA upon the addition of TtOmp85. For this experi-ment unfolded TtoA was immobilized to an ATR crystal via its His-tag. A silane-NTA linker was constructed and covalently attached to the ATR crystal. His-tagged TtoA could be immobilized to the NTA group of the linker via Ni2+. Immobilized, unfolded
6. CONCLUSION
TtoA was incubated with TtOmp85 which led to folding of TtoA into a native-like structure. The importance of TtOmp85 specically for TtoA folding was shown by several control experiments. An attempt to immobilize short TtoA peptides to the ATR crystal and to form time-stable hybrid barrels with TtOmp85 failed. All in all the technique of ATR-FTIR spectroscopy proved to be a powerful tool for the study of the insertase/substrate pair. However, in this special case TtoA has a tendency to always adopt signicant amounts of secondary structure, even at room temperature and in presence of denaturants. This makes reproducibility of the experiments di-cult. SDS has proven to be eective in keeping TtoA in a mostly unfolded state but it decreases the yield of linker-bound TtoA and does not permit the use of liposomes or other lipid environments in the experiments. For future studies, outer membrane pro-teins from a bacterium that is less thermostable than T. thermophilus might be good candidates to expand the studies on outer membrane proteins and the mechanism that is used by insertases to fold and insert substrates into membranes.
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