Inuence of denaturants on TtoA stability

To conduct folding studies on TtoA the protein had to be accessible in its unfolded state, preferably at RT. Temperature dependent measurements of TtoA showed that heat is not suited to unfold the protein. Thus, in an attempt to reduce TtoA's temperature stability, the denaturating agents urea, 2-mercaptoethanol, DTT and SDS respectively were added to native TtoA in varying concentrations. SDS-PAGEs were run to detect changes in the apparent molecular weight of TtoA and FTIR-temperature ramps in transmission mode were conducted to visualize changes in TtoA secondary structure in the second derivatives of the absorbance spectra.

5.1.3.1 Native TtoA in urea

Urea, along with guanidine hydrochloride (GdnHCl), is a strong denaturing agent and frequently used to unfold proteins. For FTIR measurements it is not convenient to use urea or GdnHCl because both strongly absorb in the amide I or amide I' region.

The application of ¹³C urea, which has a down-shifted absorbance, was tested so that the amide I' region would not be obscured by the urea signal. However, the extreme amounts of¹³C urea which would have been necessary to unfold TtoA were too expen-sive to justify its use as a denaturant. Nonetheless, it is possible to conduct unfolding studies on TtoA in urea by SDS-PAGE. In contrast to GdnHCl, urea does not leave broad smears on the gel when mixed at high concentrations with detergent solubilized protein. Native TtoA was treated with varying concentrations of urea ranging from 7 mol/L to 10 mol/L and incubated either at RT or at 100 ^◦C for 15 minutes

(Fig-5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

ure 5.5). Protein that was incubated at RT showed a band that ran slightly below 25 kDa which is typical for native TtoA. When the protein was boiled an additional band appeared. This band was upshifted to match the 25 kDa band. The 25 kDa band is common for unfolded TtoA which has a less compact structure than folded TtoA.

However, despite the highly concentrated urea in all samples, complete unfolding of TtoA was not achieved. Even 10 mol/L urea plus additional heating did not result in a sample of completely folded TtoA when cooled down to RT. It might be possible to increase urea concentration further and thus obtain unfolded TtoA. However, already the common concentration of urea that is used for unfolding of proteins (8 mol/L) is problematic for FTIR measurements because of the intense urea absorbance bands in the amide I/amide I' region of FTIR spectra. The urea signal could be minimized by the use of very small pathlengths during FTIR measurements but the protein concen-trations that would be required in order to detect secondary structure elements would have to be higher than the ones that were used within this thesis. The required urea concentrations to successfully unfold native TtoA in FC-12 buer must be higher than 10 mol/L which would result in an amide I' signal completely masking the protein signal and that can not be subtracted from the overall spectrum anymore because its absorbance is so high that the detector receives hardly any signal anymore. The use of

13C urea at concentrations required for unfolding of TtoA is nancially highly incon-venient. As a consequence, urea was not used as a denaturant in further experiments.

Figure 5.5: Native TtoA was incubated with (A) 7 M, (B) 8 M, (C) 9 M, (D) 10 M urea.

Samples marked with * were boiled at 100 ^◦C for 15 minutes

5.1.3.2 Native TtoA in 2-mercaptoethanol

TtoA shows a remarkable resistance toward high temperatures, as was established in subsection 5.1.1. This stability might be partly due to an extracellular disulde bridge because such disulde bridges are known to be important for protein folding and stability.^[194]In order to test the signicance of the extracellular disulde bridge between

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

TtoA β-strands 9 and 10 (Figure 2.6),^[93] the reducing agent 2-mercaptoethanol was added to the protein in various concentrations and temperature ramps were conducted.

In addition, SDS-PAGEs were run with native TtoA samples in varying concentrations of 2-mercaptoethanol that were either incubated at RT or at 102 ^◦C before the SDS-PAGE.

The IR spectrum of TtoA in 4% 2-mercaptoethanol (Figure 5.6) shows a splitting of the amide I' band at 20 ^◦C (dark blue spectrum) that is indicative for anti-parallel β-structure. The signicant bands of the spectrum at 20 ^◦C are at 1629 cm⁻¹ and at 1696 cm⁻¹. The band at 1629 cm⁻¹ seems to be broader than usual second derivative bands which might be due to two or more bands that are in very close proximity to one another and indicate β-sheet interaction with varying H-bond strengths. At 100 ^◦C (red, dashed spectrum) the bands at 1629 cm⁻¹ and 1696 cm⁻¹ shift position to 1630 cm⁻¹ and 1693 cm⁻¹, respectively, and decrease in intensity but do not vanish completely. However, these intensity changes indicate the beginning of an unfolding process. The cooling of the sample to room temperature shows a shift of the bands at 1630 cm⁻¹ and 1693 cm⁻¹ to 1626 cm⁻¹ and 1691 cm⁻¹ and a rise in intensity of the bands. Comparing the spectra before and after heating shows that bands at 1629 cm⁻¹ and 1626 cm⁻¹ are 3 cm⁻¹ and bands at 1696 cm⁻¹ and 1691 cm⁻¹ are 5 cm⁻¹ apart.

This structural change is small and not visible in the SDS gel sample A (Figure 5.7) because cooling the sample after boiling causes TtoA to refold. The refolded TtoA shows the same apparent molecular weight as the native protein. However, SDS-PAGE shows that at higher 2-mercaptoethanol concentrations of 5 to 7%, the sample that was boiled and cooled down to room temperature again has a visible amount of unfolded TtoA, which runs at a higher apparent molecular weight than more compact, folded TtoA.

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

Figure 5.6: Temperature ramp of TtoA in 4% 2-mercaptoethanol. The second derivatives of the absorbance spectra are shown.

Figure 5.7: SDS-PAGE of native TtoA in 2-mercaptoethanol. Native TtoA was incubated with (A) 4%, (B) 5%, (C) 6%, (D) 7% 2-mercaptoethanol. Samples marked with * were boiled at 102^◦C for 15 minutes.

In order to test the eect of even higher 2-mercaptoethanol concentrations on TtoA stability, temperature ramps of native TtoA in 10%2-mercaptoethanol were run (Fig-ure 5.8). The dark blue spectrum was recorded at 20^◦C and shows bands at 1630 cm⁻¹ and at 1695 cm⁻¹. These are assigned toβ-structured native TtoA. The band at 1630 cm⁻¹ seems to include at least two overlapping minima due to its asymmetric shape with a hinted shoulder on the right side. Heating the sample to 100^◦C (red spectrum) results in the complete disappearance of the bands at 1630 cm⁻¹ and at 1695 cm⁻¹. Complete unfolding seems to take place. In accordance with this observation, minima in a region between 1647 cm⁻¹ and 1651 cm⁻¹ appear and indicate random coil or open loop structure. Recooling the protein to 20^◦C (black dotted spectrum) results in bands at 1624 cm⁻¹ and 1686 cm⁻¹. The shift of 6 cm⁻¹ from 1630 cm⁻¹ to 1624 cm⁻¹ and 9 cm⁻¹ from 1695 cm⁻¹ to 1686 cm⁻¹ suggests that refolding to a non-native structure has taken place. To test whether further structural changes could be induced, the

sam-5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

ple was reheated to 100^◦C. The result is again complete unfolding ofβ-structure (pink spectrum). Recooling of the sample (green dashed spectrum) results in bands that are at positions 1624 cm⁻¹ and 1686 cm⁻¹. Thus, no further structural change has taken place.

Figure 5.8: Temperature-dependent FTIR-spectra of native TtoA in 10 % 2-mercaptoethanol. The second derivatives of the absorbance spectra are shown.

In contrast to SDS-PAGE, FTIR spectra do not suggest that part of the TtoA sample remains unfolded after recooling to room temperature. An explanation for this circum-stance might be that TtoA of a less compact structure, which runs at higher apparent molecular weight than native TtoA, does not necessarily have to be in a completely unfolded state. The residual secondary structure that is still present in unfolded TtoA shows as bands in the amide I' region of FTIR spectra. Thus, it might be concluded that 2-mercaptoethanol is able to eciently unfold TtoA at 100^◦C but does not keep it suciently unfolded when the sample cools to room temperature. The extracellular disulde bridge of TtoA seems to contribute strongly to the protein's stability towards heat and its ability to fold into a native-like conformation. Changes in the mid-amide I' region where various other structural elements absorb are too small for a reliable interpretation of changes in secondary structure elements such as α-helix or random coil.

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

5.1.3.3 Native TtoA in dithiothreitol

In order to conrm that the reduction of the disulde bridge is the cause of decreased TtoA temperature stability, experiments were repeated with dithiothreitol (DTT).

DTT, like 2-mercaptoethanol, reduces disulde bridges and thus should have the same destabilizing eect on TtoA stability. SDS-PAGES on native TtoA were run at varying concentrations of DTT, ranging from 100 mM to 400 mM (Figure 5.9). The samples were either kept at RT or boiled at 100^◦C before the SDS-PAGE. 100 mM and 200 mM DTT in combination with boiling (line A* and B*, respectively) did not result in a shift of the TtoA band to a higher apparent molecular weigth. At 300 mM DTT and boiling, the TtoA band splits into two components which indicates the presence of unfolded and folded TtoA (line C*). The addition of 400 mM DTT to native TtoA and subsequent boiling has the same eect (line D*).

Figure 5.9: SDS PAGE of native TtoA incubated with (A) 100 mM, (B) 200 mM, (C) 300 mM, (D) 400 mM DTT. Samples marked with * were boiled at 102^◦C for 15 minutes. An upward shift of the TtoA band is due to a less compact structure and therefore a higher apparent molecular weight.

Temperature ramps of native TtoA were run at a concentration of 400 mM DTT (Figure 5.10). At 20 ^◦C the spectrum shows bands at 1628 cm⁻¹ and 1695 cm⁻¹. At 100 ^◦C the intensity of these bands decreases signicantly but they do not vanish completely. After cooling the bands reappear with a downshift to 1625 cm⁻¹ and 1688 cm⁻¹. Partial unfolding takes place in presence of the reducing agent DTT, and refolding to a denitely native structure does not take place, although signicant amounts of β-structure reform. The TR was repeated with 500 mM DTT, yielding a similar result. At 20 ^◦C the spectrum shows bands at 1629 cm⁻¹ and 1695 cm⁻¹. Heating does not induce complete unfolding of TtoA. The amide I' bands are still clearly visible at 100 ^◦C. After recooling, the bands shift to 1625 cm⁻¹ and 1686 cm⁻¹. Both

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

DTT and 2-mercaptoethanol seem to destabilize TtoA and hinder correct refolding, which is likely due to the loss of the extracellular disulde bridge (Figure 2.6).

Figure 5.10: Temperature-dependent FTIR spectra of native TtoA in DTT. Second deriva-tives are shown. Native TtoA was heated from 20 ^◦C to 100 ^◦C and sub-sequently recooled to 20 ^◦C in presence of 400 mM DTT (upper panel) and 500 mM DTT (lower panel). Before the TR at 20 ^◦C the spectra show a splitting of the amide I' band that is due toβ-structure (blue). For both DTT concentrations heating to 100^◦C caused a decrease inβ-structure (red). Sub-sequent cooling led to reformation ofβ-structure (black), but the minima are downshifted in relation to native TtoA before heating.

5.1.3.4 Native TtoA in SDS

To conduct folding studies on TtoA the protein had to be accessible in its unfolded state, preferably at room temperature. The widely used denaturants urea, guanidine hydrochloride and 2-mercaptoethanol did not prove to be ecient enough for TtoA unfolding. To test another denaturant, the ionic detergent SDS, which is known to break up protein secondary structure by covering the protein backbone with negative

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

charges, was tested because it does not show signicant absorbance in the amide I region of FTIR spectra. For a spectrum of SDS see Figure 4.4.

SDS-PAGEs were run on native TtoA that was incubated with dierent concentrations of SDS, ranging from 0.5 % to 2 % w/v (Figure 5.11). The samples were either incu-bated at RT before the SDS-PAGE or boiled at 102^◦C for 15 minutes. The addition of SDS to native TtoA did not cause shifts in TtoA's apparent molecular weight. How-ever, additional boiling of the samples rendered increasing amounts of unfolded protein with increasing SDS concentration. The new band runs at a higher apparent molecular weight of 25 kDa than the band of native TtoA, indicating a less compact structure.

Even the concentration of ten times SDS's CMC (0.2%) is apparently not enough to achieve signicant unfolding of TtoA.

Figure 5.11: SDS PAGE of TtoA in SDS. Native TtoA was incubated with (A) 0.5%, (B) 1%, (C) 1.5%, (D) 2%SDS. Samples marked with * were boiled at 100 ^◦C for 15 minutes.

Although temperature ramps in presence of 2 % SDS (Figure 5.12) show that slight unfolding of native TtoA (blue spectrum) takes place at 100 ^◦C (red spectrum), only a little fraction of the protein remains unfolded after recooling of the sample to room temperature (black spectrum). A shift of the amide I' bands takes place between native TtoA and recooled TtoA. The low-frequency band shifts from 1629 cm⁻¹ to 1626 cm⁻¹ and the high frequency band shifts from 1695 cm⁻¹ to 1693 cm⁻¹. Despite this spectral change, induced by boiling in SDS, the protein shows the same running behavior as native TtoA on SDS gels up to concentrations of 2 %SDS.

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

Figure 5.12: Temperature-dependent FTIR spectra of native TtoA in 2%SDS. Native TtoA was heated to 100^◦C in presence of 2%SDS. Second derivatives of absorbance spectra are shown.

Native TtoA was puried from T. thermophilus in buer with 0.1 % FC-12, a non-ionic detergent that keeps the protein soluble outside a lipid bilayer. The girdle of FC-12 micelles around TtoA might be the reason why SDS can only insuciently attack TtoA secondary structure and cause unfolding. To test this assumption TtoA was puried from E.coli inclusion bodies in presence of SDS instead of FC-12 (see subsubsection 5.1.3.6).

5.1.3.5 Native TtoA in 2-mercaptoethanol and SDS

In order to test the inuence of a combination of denaturants on the temperature stability, 2 % SDS and 4 % 2-mercaptoethanol were added to native TtoA before heating the sample to 100 ^◦C (Figure 5.13). At 20 ^◦C the split bands of the amide I' signal at 1628 cm⁻¹ and 1696 cm⁻¹ indicate the presence of β-structure. The bands disappear at 100 ^◦C (Figure 5.13, red line) and reappear after cooling the sample to 20^◦C at 1624 cm⁻¹ and 1686 cm⁻¹. The slight downshift indicated non-native but not aggregated β-structure. The combination of two denaturants at high concentrations thus was not successful in keeping TtoA unfolded at RT.

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

Figure 5.13: Native TtoA in 2%SDS and 4%2-mercaptoethanol was heated from 20^◦C to 100^◦C and then recooled to 20^◦C. Complete unfolding was not achieved, and amide I' bands are shifted to slightly lower wavenumbers after the TR. Second derivatives of the absorbance spectra are shown.

5.1.3.6 TtoA from inclusion bodies in SDS

The previous sections showed that no solution could be found to keep native TtoA from T.thermophilus puried in FC-12 in an unfolded state at room temperature. However, the protein can also be expressed as inclusion bodies in E.coli and puried in the presence of SDS instead of FC-12. Figure 5.14 shows an SDS-PAGE which was run on TtoA puried from T. thermophilus in 0.1 %FC-12 (A) and from E. coli in 0.4 % SDS (B), respectively. The puried protein was either incubated at RT before the SDS PAGE or boiled at 102^◦C for 15 minutes. The whole native TtoA sample runs at one apparent molecular weight (A) which does not shift after boiling (A*). TtoA that was puried from E. coli inclusion bodies and incubated at RT displays two distinct bands on the SDS gel (B). The lower band indicates TtoA with an apparent molecular weight that is very similar to that of native TtoA. The upper band is at a slightly higher apparent molecular weight which is assigned to a less compact structure. When the sample is boiled (B*) the lower band almost completely vanishes and the upper band grows more intense. Almost complete unfolding of TtoA seems to take place.

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

Figure 5.14: TtoA from T.thermophilus before (A) and after (A*) boiling at 102^◦C. The running behavior of both samples is the same. Both, folded and unfolded TtoA is present in samples from inclusion bodies before boiling (B). After boiling (B*) almost all TtoA is unfolded.

The results from SDS-PAGE were compared to temperature ramps of TtoA from in-clusion bodies (Figure 5.15). The upper panel shows a second derivative spectrum at 20 ^◦C (blue) that has signicant minima at 1624 cm⁻¹ and 1684 cm⁻¹. These corre-spond to anti-parallel β-structure. However, they are shifted to lower wavenumbers compared to native TtoA (Figure 5.2). These lower-frequency minima are comparable to those that are visible after native TtoA in 2-mercaptoethanol or DTT is heated to 100 ^◦C and recooled to 20 ^◦C (subsubsection 5.1.3.2, subsubsection 5.1.3.3). Heating the sample to 100^◦C (red) results in a signicant reduction of the 1624 cm⁻¹ band, but it is still visible. The intensity does not change signicantly after cooling of the sample to 20 ^◦C (black) and band positions remain the same. The same TtoA from inclusion bodies was heated to 110 ^◦C (Figure 5.15, lower panel). At 20 ^◦C (blue) the second derivative spectrum shows minima at 1624 cm⁻¹ and 1684 cm⁻¹. Heating to 110 ^◦C (red) results in a complete disappearance of the 1624 cm⁻¹ band. It does not reappear after cooling the sample to 20^◦C. Here, too, the 1684 cm⁻¹ is mostly unaected by the TR.

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

Figure 5.15: TtoA from E. coli inclusion bodies was heated to 100 ^◦C (upper panel) and to 110 ^◦C (lower panel). At 20 ^◦C both samples showed intense bands at 1624 cm⁻¹and 1684 cm⁻¹. At 100^◦C the bands have almost vanished. After recooling to 20 ^◦C there are still remnants of the bands visible. At 110 ^◦C the bands disappear completely. Recooling to 20 ^◦C does not lead to the reappearance of these bands but formation of a lower-frequency band.

SDS-PAGE of TtoA samples from inclusion bodies were run to show that TtoA remains unfolded for at least 24 hours (Figure 5.16). Panel A shows TtoA from inclusion bodies that was incubated at room temperature for 24 hours. The sample shown in panel B was boiled at 102 ^◦C for 15 minutes and then left at RT for 24 hours. Sample C was incubated at RT for 24 hours and then boiled at 102 ^◦C for 15 minutes. The unboiled sample A runs at an apparent molecular weight of slightly below 25 kDa. The boiled samples B and C both have upshifted apparent molecular weights at about 25 kDa.

The upshift indicates a less compact conformation. However, secondary structures may still remain, and TtoA is able to formβ-structure after some time, even when dissolved in SDS. Thus, for folding experiments, TtoA from E.coli inclusion bodies was boiled for

5. RESULTS AND DISCUSSION 5.1. STABILITY OF TTOA AND TTOMP85

fteen minutes directly before the experiment, and changes in secondary structure were monitored closely after initiation of the folding process. These changes were compared to structural changes that took place without initiation of the folding process. For details on the folding experiments refer to section 5.2

Figure 5.16: TtoA from E. coli inclusion bodies was (A) incubated at RT for 24 h or (B) incubated at 102^◦C for 15 min and then at RT for 24 h or (C) incubated at RT for 24 h and then at 102^◦C for 15 min.