Temperature stability of native TtoA in detergent

In order to test the stability of TtoA towards high temperatures, SDS-PAGEs of un-boiled and un-boiled TtoA were run (see Figure 5.1). Native TtoA was stored in Tris-HCl buer pD 7.4 with the non-ionic detergent Fos-choline 12 (FC-12). FC-12 was in the FC-12 buer 0.1 % w/v (which is twice its critical micellar concentration (CMC) of 0.0.5% w/v) in order to keep TtoA soluble outside its native membrane environment.

TtoA was kept at RT for 15 minutes before the SDS loading buer was added and the SDS PAGE was run (A). The protein shows an apparent molecular weight that lies just below the 25 kDA marker band and corresponds to TtoA's molecular weight of 23 kDa. The apparent molecular weight and thus the compact structure of TtoA is not aected when incubated at 102 ^◦C for 15 minutes (A*).

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

Figure 5.1: SDS PAGE of TtoA puried from T. thermophilus. The sample that was incu-bated at RT (A) runs at the same apparent molecular weight as the sample that was incubated at 102^◦C (A*)

SDS-PAGE allows to detect shifts in the apparent molecular weight of TtoA, which is due to a less compact structure. The structure of native TtoA seems unaected by temperatures up to 100 ^◦C. To detect possible small changes in TtoA structure that do not eect the protein's apparent molecular weight, temperature-dependent FTIR spectra were recorded. Measurements were conducted in transmission mode, involving the heating of the protein from 20^◦C to 100^◦C and the subsequent recooling to 20 ^◦C.

For the detailed procedure, refer to chapter 4. Second derivatives of the recorded absorbance spectra were compared in order to follow any changes in secondary structure during and after the process.

Figure 5.2 a) shows absorbance spectra of native TtoA that have two prominent bands at 1628 cm⁻¹ and 1695 cm⁻¹. The second derivative was used to assign these bands in the dark blue spectrum at 20 ^◦C before the temperature ramp (TR) to 1628 cm⁻¹ and 1695 cm⁻¹ (Figure 5.2 b)). These bands represent the split signal that is as-signed to anti-parallel β-strands and consists of a weak high-frequency and an intense low-frequency band. The band maximum shifts during the TR in a region between 1627 cm⁻¹ and 1632 cm⁻¹. The shift is attributed to the inuence of heat on the strength of the hydrogen bonds that stabilize theβ-structure. However, this frequency change is not signicant enough to conclude a major structural change in theβ-barrel.

The existence of dierent hydrogen bond strengths within TtoA β-structure might

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

also be reected in the presence of a shoulder at 1624 cm⁻¹ which becomes visible at 100^◦C and remains visible after cooling Figure 5.2. Due to the presence of a dual low-frequency signal in native TtoA that is immobilized in H2O to an IRE for ATR-FTIR spectroscopy (subsection 5.2.2) this shoulder probably does not indicate a structural change within the β-barrel of TtoA into non-native conformations. The eect on the solvent D2O on TtoA spectra might play a role regarding the degree of detail in which theβ-structure bands are resolved. Thus, the assumption is that native TtoA in FC-12 buer is not sensitive to heat, even at 100^◦C.

While the major bands in Figure 5.2 were assigned toβ-structure, smaller bands within the amide I' region are visible in the second derivative as well, and these are assigned to heterogeneous secondary structure such as the two α-helices, fourteen β-turns and sevenβ-hairpins that were identied in the TtoA crystal structure.^[93]The extracellular α-helices, for example, are not part of the thermostable TtoA transmembrane barrel and might thus be aected by high temperatures, as uctuations in band positions and intensities in the amide I' region during the TR might indicate. However, it is dicult to dierentiate between these minor bands and the structures they represent.

Nonetheless, TtoA temperature stability is obvious from the TR. The choice of non-ionic detergent does not seem to have an inuence on TtoA stability. For a TR of native TtoA in the non-ionic detergent n-Octyltetraoxyethylene (C8E4), see Figure 5.3. A direct comparison of TtoA in FC-12 and TtoA in C8E4 shows no signicant dierences in temperature resistance. The amide I' bands at 1630 cm⁻¹ and 1693 cm⁻¹ are mostly unaected by heat. The minor shift of the 1693 cm⁻¹ and the shoulder of the low-frequency band may be due to dierent H-bond distances in the β-structures and the presence of tightly packed and weakly interacting β-sheets.^[191]

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

Figure 5.2: IR spectra of native TtoA in dependence of temperature. Left: absorbance spectra. Right: second derivatives of absorbance spectra. The IR spectrum shows ngerprint bands at 1628 cm⁻¹and 1695 cm⁻¹that indicate anti-parallel β-structure. The band positions and intensities do not change signicantly during heating and after cooling of the sample. A shoulder around 1624 cm⁻¹becomes visible after heating but is not accredited to major structural changes.

Figure 5.3: Temperature ramp of native TtoA in 0.35 % of the noionic detergent n-Octyltetraoxyethylene (C8E4). Second derivatives of the absorbance spectra are shown. The TR starts at 25^◦C and moves in steps of 5^◦C to 100^◦C before cooling back down to 20^◦C. The minimum at 1630 cm⁻¹does not shift. Instead, a small shoulder appears at slightly lower wavenumbers that is not accredited to major structural changes. The minimum at 1693 cm⁻¹ shifts to slightly lower wavenumbers but does not indicate signicant structural change.

A high temperature resistance is common in Omps.^[192] However, for aβ-barrel protein in detergent micelles, TtoA shows a remarkable thermostability even compared to other

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

Omps. This becomes clear when TtoA TR spectra are directly compared to those of TtOmp85, another Omp from the same thermophilic organism (see subsection 5.1.2).

Stabilizing forces within the TtoAβ-barrel might cause the temperature resistance, as well as an extracellular disulde bridge between sheets 9 and 10 (Figure 2.6). This hypothesis is tested in subsubsection 5.1.3.2 and leads to the assumption that the disulde bridge is indeed a highly stabilizing factor for TtoA.