TtOmp85-assisted folding of TtoA

In order to record the eect of TtOmp85 on TtoA folding, small concentrations of TtOmp85 were added to the unfolded, His-tagged, linker-immobilized TtoA in SDS buer which is described in chapter 4 (Figure 5.24).

Figure 5.24: TtOmp85-assisted folding of TtoA. Unfolded TtoA was immobilized to the IRE with silane-NTA linker molecules. ATR spectra were measured for 120 min-utes after addition of TtOmp85. TtOmp85-induced conformational changes of TtoA from an unfolded structure (blue) to the native-likeβ-sheet structure (red) are observed.

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

Figure 5.25: As a control for TtOmp85-induced folding of TtoA, unfolded TtoA was incu-bated with the same volume of FC-12 buer (see chapter 4) as in Figure 5.24 but in absence of TtOmp85. The concentration of FC-12 was 0.01%i.e. below the CMC of 0.05%. This shows that FC-12 at this concentration is not able to initiate folding of TtoA.

TtOmp85 in FC-12 buer (for details on buer composition see chapter 4) was di-rectly pipetted onto the crystal. The nal TtOmp85 concentration on the IRE was 0.3 mg/mL. The nal buer composition contained almost 0.4%SDS (CMC = 0.2 % w/v) but only 0.01%FC-12 (CMC = 0.05%w/v). The CMC of FC-12 was not reached and FC-12 induced folding of TtoA was not expected.^[182] Spectra were recorded every 60 seconds to monitor changes in TtoA secondary structure. Changes in the amide I region were recorded over a course of two hours. TtOmp85 was then rinsed o the crys-tal with SDS buer (for details on buer composition see chapter 4). TtOmp85 itself does not contribute to the spectra because of the high local TtoA concentration on the crystal surface compared with the resulting small overall concentration of TtOmp85 (3.5 µM or 0.3 mg/mL). Figure 5.24 shows the results of the incubation of unfolded TtoA with TtOmp85. The main band at 1649 cm⁻¹, minor bands at 1697 cm⁻¹ and 1682 cm⁻¹ and a shoulder at 1634 cm⁻¹ disappear with time. Instead, a split signal at 1629 cm⁻¹, 1638 cm⁻¹ and 1689 cm⁻¹ appears which has already been assigned to TtoA native structure, and where the band at 1638 cm⁻¹ might hint at a less tightly packedβ-structure than the band at 1629 cm⁻¹.^[191] A band at 1657 cm⁻¹ appears and is assigned to various structure elements including α-helices and β-turns. In contrast,

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

when the same volume of FC-12 buer without TtOmp85 is added to unfolded TtoA, no bands indicating structural changes were monitored (Figure 5.25).

The interaction between SDS-solubilized TtoA and TtOmp85 that was kept in FC-12 buer and is transferred into SDS buer should only be possible if the FC-12 belt around TtOmp85 remains intact. Should an exchange for SDS monomers take place, the interaction between TtOmp85 and TtoA should be prevented due to strong negative charges around both proteins. Therefore we assume that either TtOmp85 remains in FC-12 micelles or that TtoA is stripped of SDS upon interaction with TtOmp85. It should be mentioned that TtoA folding could be initiated when concentration of FC-12 reached the CMC. An exchange of 50%of SDS buer, in which the immobilized TtoA was kept, for 50% of FC-12 buer started a folding process (Figure 5.26).

Figure 5.26: Incubation of unfolded TtoA with FC-12 as control. 50%of SDS buer (SDS buer) were exchanged for FC-12 buer (FC-12 buer) yielding a concentration of 0.2%SDS and 0.05%FC-12. These concentrations correspond to the CMCs of the respective detergents (SDS = 0.2%w/v and FC-12 = 0.05%w/v). The blue spectrum shows unfolded TtoA. The green and red spectra were recorded 10 minutes and respectively 50 minutes after dilution with SDS buer. The red spectrum shows refolded TtoA. The experiment demonstrates that TtoA can refold in non-ionic detergent FC-12 at the CMC.

The major changes in band positions and intensities are shown over a time-course of 60 minutes. The band at 1648 cm⁻¹ of TtoA in SDS buer is assigned to 'open-loop' or random coil structure. 'Open-'open-loop' structure might be present due to amino

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

acid stretches without peptide backbone interactions and its position was assigned to circa 1644 cm⁻¹ by Fabian et al.^[160] Coil structures might be present in the SDS solubilized state. Residual β-structure is visible in the spectrum of unfolded TtoA with two high-frequency components (at 1681 cm⁻¹ and 1695 cm⁻¹) but only one low-frequency contribution at 1633 cm⁻¹. The band at 1648 cm⁻¹ disappears after addition of FC-12 and a band at 1654 cm⁻¹ appears during the incubation time (Figure 5.26, red spectrum). This band is assigned to the presence of heterogeneous secondary structure including α-helices and coils. After incubation with FC-12 the structure shows two low-frequency bands at 1633 cm⁻¹ and 1629 cm⁻¹ and one high-frequeny band at 1687 cm⁻¹for theβ-structure. The additional low-frequency band at 1629 cm⁻¹ along with the shifted high-frequency band at 1687 cm⁻¹ might be due to more tightly packed β-structure.^[191] Since the FC-12 concentration was kept below the CMC in the experiments with TtOmp85 we conclude that it is the addition of TtOmp85 that initiates folding of TtoA in SDS and not FC-12.

To test whether TtoA needs specically TtOmp85 to fold, we conducted a control ex-periment in which we immobilized unfolded TtoA in SDS buer and, instead of adding TtOmp85 in FC-12 buer, we added native TtoA in FC-12 buer to initiate folding (Figure 5.27). In the spectrum of immobilized, unfolded TtoA in 0.4 % SDS, weak components at 1628 cm⁻¹ and 1688 cm⁻¹ indicate residual β-structure which is always present in TtoA due to its tendency to formβ-structure even at high concentrations of SDS and after boiling.^[175] The splitting of the high frequency band is not visible here as it is in Figure 5.24 and Figure 5.26 (blue spectra) but we assume that TtoA is in a similar unfolded conformation due to only one visible low-frequency band (1628 cm⁻¹).

Spectra were recorded after addition of 0.6 mg/mL TtoA in FC-12 buer, in which the FC-12 concentration was 0.01 % and below the CMC of 0.05 %. The blue spectrum shows unfolded TtoA 10 minutes after the addition of native TtoA. No major struc-tural changes were detected. 120 minutes after addition of native TtoA we still did not detect structural changes in the immobilized TtoA. Thus we conclude that folding of TtoA can only be initiated by TtOmp85 and not by anotherβ-barrel protein.

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

Figure 5.27: Incubation of unfolded TtoA with native TtoA instead of TtOmp85 as a con-trol. Unfolded, immobilized TtoA was incubated with 0.6 mg/mL native TtoA in FC-12 buer. The nal concentration of FC-12 buer in the sample was approximately 0.01%i.e. below the CMC (0.05% w/v). Absorbance spectra of unfolded TtoA in 0.4 % SDS (black spectrum), unfolded TtoA after 10 minutes of incubation with native TtoA (blue spectrum) and after 120 minutes (red spectrum) are shown. No signicant structural changes take place during incubation with native TtoA indicating that the assisted folding is TtOmp85-specic.

It should be mentioned that, in all experiments that involved unfolded TtoA, the stability of the Omp towards SDS remained problematic, even when TtoA was puried from inclusion bodies. Only freshly puried samples could be unfolded by heat, as was shown on SDS-PAGE Figure 5.30. As can be seen in most spectra of immobilized TtoA from inclusion bodies, signicant amounts of secondary structure remain. The older the sample, the more secondary structure is visible in the FTIR spectra, so that individual folding experiments start with unfolded TtoA that shows dierent amounts of secondary structure. SDS-PAGE showed that in case of older TtoA samples the protein ran at an apparent molecular weight of native TtoA and it could not be unfolded by boiling. The consequence of TtoA's tendency to form secondary structure even in SDS is the limited reproducibility of folding experiments since we cannot assume that unfolded, immobilized TtoA shows the same degree of unfolding in every experiment.

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

For further, more detailed studies of Omp folding experiments it will be necessary to completely destabilize TtoA or continue investigations on a dierent protein.

The time-dependent folding of TtoA folding initiated by TtOmp85 is shown in Fig-ure 5.28. However, these results are not quantitatively reliable because of several reasons. Firstly, the experimental setup is not automated. This means that the delay time before the rst measurement can vary signicantly, ranging from 5 to 30 seconds, approximately. Further, the amount of immobilized TtoA can not be controlled in this experiment. The concentration depends on the success rate of linker immobilization, on the addition of NTA-groups to the isocyanate group and on the binding of TtoA to the NTA groups. Also, the numerous pipetting steps during the experiments might lead to variations in the sample volume on the crystal and thus to slightly dierent TtOmp85 concentrations, which would inuence the reaction kinetics. TtOmp85 activity was not tested after purication of the protein, thus the total concentration of TtOmp85 in the sample might dier from the concentration of active molecules. Further, the amount of residual secondary structure of immobilized TtoA diers for each experiment due to reasons that were explained in the previous paragraph. It must also be mentioned that, due to dierences in the intensity of the background spectrum, β-sheet bands might show uctuations in intensity although they overall increase in comparison with random coil bands Figure 5.29. The spectral bands are marked with 1650 cm⁻¹ and 1638 cm⁻¹ for easier comparison with Figure 5.28, upper panel. However, the actual positions dier by 1 cm⁻¹ - 2 cm⁻¹. Thus, comparing dierent folding times with each other will not lead to reliable results.

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

Figure 5.28: The upper panel shows spectra that were recorded during the incubation of TtoA with TtOmp85 at dierent time points. The second panel depicts the decay of the 1650 cm⁻¹ band which is assigned to unfolded or 'open loop' structure. Data points begin at 5 minutes after addition of TtOmp85 due to equilibration time and t1 is 16.485 min.The lower panel shows the rise of the band at 1638 cm⁻¹. Data points start at 5 minutes after addition of TtOmp85

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

Figure 5.29: Repetition of the experiment depicted in Figure 5.28 shows that reproducibility is limited.

Figure 5.30: SDS PAGE of TtoA from inclusion bodies. Samples marked with an asterisk were heated to 102 ^◦C before the experiment. The unfolding of TtoA from inclusion bodies is only successful in fresh samples.

5.2.3.1 Spectra of native, unfolded and refolded TtoA

Figure 5.31 shows a direct comparison of ATR spectra from native, unfolded and re-folded TtoA. We distinguish between native and rere-folded TtoA because native TtoA was puried from T. thermophilus and immobilized in its native conformation whereas refolded TtoA was puried from E. coli inclusion bodies and immobilized in its unfolded conformation and refolding only occurred upon addition of TtOmp85 (Figure 5.24). A good match in band positions of native (black spectrum) and refolded TtoA (blue

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

trum) is observed. The split signal at 1629 cm⁻¹, 1638 cm⁻¹ and 1686 cm⁻¹ is assigned toβâstructure. The band at 1659 cm⁻¹ in the native TtoA indicates the presence of α-helical structure, β-turns and loops, which are all likely to absorb in approximately the same spectral region. The 'open-loop' or coil band at 1648 cm⁻¹ that is apparent in the spectrum of unfolded TtoA (Figure 5.31, red) has disappeared after TtOmp85 incubation (Figure 5.31, blue). TtoA seems to refold into a native-like conformation because the spectra of native and refolded TtoA are very similar. The refolding took place in SDS buer which contained 0.4 % SDS. It is well known that β-barrels are very stable at high SDS concentrations which might explain TtoA's ability to fold in 0.4 %SDS with the help of TtOmp85.[175,176,198]

Figure 5.31: Comparison of native, unfolded and refolded TtoA. ATR-FTIR spectra of im-mobilized native TtoA (black), unfolded TtoA (red) and refolded TtoA (blue) are shown. The spectrum of refolded TtoA was recorded after the incubation with TtOmp85 for two hours and subsequent rinsing of the IRE to remove TtOmp85. The spectroscopic approach proves that TtOmp85-assisted refold-ing of TtoA ends in a native-like conformation.