Summary about the role of TtOmp85 in TtoA folding

5.2.4.1 TtOmp85-assisted folding of TtoA in presence of SDS

The partners TtOmp85/TtoA were chosen for this study because they represent a model of the insertase/substrate system which acts in the absence of any accessory

5. RESULTS AND DISCUSSION 5.2. ROLE OF TTOMP85 IN TTOA FOLDING

proteins that might be important for TtOmp85 function. This simplicity distinguishes Thermus TtoA insertion and folding from the BAM system in E. coli which, in addition to the Omp85 homolog BamA, contains four accessory BAM proteins. ATR experi-ments were performed monitoring changes in secondary structure of immobilized TtoA upon interaction with TtOmp85. The ATR spectra of refolded TtoA after the interac-tion with TtOmp85 showed bands corresponding to β-structure matching those seen in the native protein. Thus, unfolded TtoA was able to fold into a native-like structure when TtOmp85 was present. In contrast to other folding studies of Omps the denatu-rant was not diluted in order to initiate folding.^[8084] The use of SDS as a denaturant instead of the more commonly used agents urea or guanidine hydrochloride resulted from previous TtoA unfolding experiments^[175] which are described in subsection 5.1.3.

These experiments showed that extremely high concentrations of these denaturants in combination with temperatures of 100^◦ C or higher are required to achieve unfolding of native TtoA. Urea and guanidine hydrochloride absorb strongly in the amide I region and hinder reliable subtraction of the absorbance bands from the protein spectra. On the other hand, SDS does not interfere with the protein signal in the amide I region.

Although TtOmp85 is added to SDS buer (0.4%SDS) for the folding reaction of TtoA it could be that the TtOmp85 structure remains intact because it is known that many outer membrane proteins are very resistant to SDS at room temperature.[14,175,188,199]

TtoA refolding was successfully initiated in SDS as seen from the formation of β -structure although interaction of TtOmp85 and TtoA in principle could be hindered by the negative charges of the ionic detergent. It is possible that there is no exchange of FC-12 for SDS after addition of TtOmp85 to SDS buer or that TtoA is stripped of SDS upon interaction with TtOmp85. Thus, SDS was the preferred denaturant for our experiments.

Folded and unfolded Omps dier in their apparent molecular weight in SDS PAGE^[188]

and in their line shapes in CD spectra.^[170,182]SDS-PAGE analysis showed that TtOmp85-containing proteoliposomes, which were incubated with unfolded TtoA, promoted com-pact TtoA structures which ran at higher apparent molecular weight than unfolded TtoA.^[100] However, it is not clear from these methods if the folded Omp really adopts

5. RESULTS AND DISCUSSION 5.2. ROLE OF TTOMP85 IN TTOA FOLDING

a homogenous conformation which is close to the native one, because these methods are not sensitive enough to detect structural details. On the other hand, FTIR spec-troscopy can dierentiate between structural elements such as turns, loops or intra-and intermolecular β-structure, as for example Korkmaz et al. have shown for pH dependent dierences in OmpG structure.^[191]

A test showed that native TtoA cannot initiateβ-structure formation in unfolded TtoA.

This demonstrates that TtOmp85 possesses specic chaperone functionality forβ-sheet formation which is independent of the presence of a bilayer membrane. Furthermore, when FC-12 alone was added to SDS-solubilized, unfolded TtoA at a concentration below the CMC it cannot initiate folding of TtoA. This experiment served as a further negative control for our TtOmp85/TtoA folding study because it has repeatedly been reported before that Omps can fold in non-ionic detergent upon dilution of denaturant.

However, when FC-12 is added at a concentration corresponding to its CMC, it is observable that TtoA folding is initiated. This result corresponds to ndings that non-ionic detergent, which binds to protein as micelles, needs to be present at the CMC or above.^[182]

Our experiments show that TtoA in 0.4 % SDS only forms β-structure in presence of TtOmp85. There is little dierence in the spectra of native TtoA and refolded TtoA after removal of TtOmp85. We assume that TtOmp85 only briey forms hybrid barrels with TtoA during interaction and is released right after completion of the folding pro-cess. The interaction-time between a TtoA and a TtOmp85 molecule seems to be very short, and the current experimental setup does not allow an observation of the TtoA-TtOmp85 complex. Estrada et al.^[100] found evidence for stable hybrid transmembrane barrels of TtOmp85 when incubated with short TtoA fragments, which resulted in long-living ion channels with higher conductance levels for TtOmp85 alone. In accordance with our results, these experiments also failed to detect channels when TtOmp85 was incubated with full-length TtoA. Another indication for the short lifetime of interac-tion between TtOmp85 and full-length TtoA is that the TtoA concentrainterac-tion within the penetration layer of the evanescent wave is presumably much higher than the concen-tration of TtOmp85 in the bulk solution that covers the IRE (which is only 0.3 mg/mL

5. RESULTS AND DISCUSSION 5.2. ROLE OF TTOMP85 IN TTOA FOLDING

(3.5 µmol/l)). TtOmp85 thus seems to diuse to the immobilized TtoA molecules to initiate folding of all TtoA molecules within a time frame of 120 minutes.

5.2.4.2 Proposed interaction of TtOmp85 and unfolded TtoA

Our results can be used to compare existing models of Omp85-assisted folding of OM proteins. According to one model, the TtOmp85/TtoA interaction is via a TtOmp85 multimer which forms a pore required for TtoA folding. In absence of a membrane, it would be impossible for the protein to enter such a pore and leave it laterally. In our experiment, all existing evidence hints at a stoichiometry of one TtOmp85 per one TtoA during folding initiation. Furthermore, the lack of an Omp85 multimeric pore did not hinder TtoAβ-sheet formation. A further model suggests that Omp insertion into the OM depends on the presence of an interface between the membrane and Omp85.

The absence of such an interface in our experiments indicates that TtoA β-structure formation is not dependent on it, although the existence of such an interface might be crucial during the subsequent insertion of TtoA into the OM.^[200]A model that proposes transport of an Omp through the Omp85 monomer pore to the extracellular space and subsequent membrane insertion is not supported by our results, as our experiments were conducted in detergent. Folding initiation thus does not depend on translocation of TtoA to the extracellular space. Our observations rather suggest that TtOmp85 alone, i.e. without contribution of a lipid bilayer membrane, is able to catalyze β-structure formation. Our ndings suggest that the interaction of TtoA and TtOmp85 takes place via short-lived complexes which are in accord with the β-augmentation model.^[100] In this model for TtoA insertion the TtOmp85β-barrel is gradually enlarged as it forms a hybrid barrel with the unfolded substrate and this occurs by the interaction of the two proteins alone. It also ts with our results that TtOmp85 would approach unfolded TtoA via POTRA domains,^[100]then open laterally to allow insertion of TtoAβ-strands and subsequent enlargement of the hybrid barrel (Figure 2.5).^[126]

5. RESULTS AND DISCUSSION 5.2. ROLE OF TTOMP85 IN TTOA FOLDING

5.2.4.3 Possible applications of the ATR-FTIR setup in the study of outer membrane protein folding

The setup allows access to the sample during the experiment and the exchange of pro-tein or buer components is quick and ecient. The required propro-tein concentrations are small compared to transmission experiments, and although H2O absorbance in the amide I region remains problematic the ne-tuning of the experiment yields valuable spectral information on the formation of secondary structural elements within the in-vestigated protein. All experimental steps are performed directly on the crystal with aqueous buers as solvent. The stability of TtoA towards denaturants is still a set-back to the experiments because this Omp tends to show residual structure in its SDS bound state and complicates folding experiments. Disruption of the TtoA extracel-lular disulde bridge leads to decreased temperature stability (data not shown), and TtoA cystein mutants might be a good alternative to native TtoA in folding studies.

It should be possible to add TtOmp85 in nanodiscs to unfolded TtoA, or to study the eect of possible periplasmic chaperones on TtoA solubility and interaction with TtOmp85.^[201,202]There are yet many Omp85/substrate interactions to explore and our setup might help to shed light on the details of the insertion mechanism of Omps into the OM.