Functionalization of the IRE surface and immobilization of His-tagged proteinof His-tagged protein

The construction of molecules linking nitrilotriacetic acid (NTA) groups to various surfaces for subsequent binding of His-tagged proteins was performed previously by other groups. For example, Ataka et al. attached thiobis(succinimidylpropionate) co-valently to a gold surface and added an NTA head group.^[164]Smith et al. synthesized a

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

germanium-bound linker, also with an NTA head group.^[195] Alternatively it is possible to cover a crystal surface with a lipid lm that contains modied lipids with NTA head groups.^[166] However, working with lipids excludes the use of SDS or other detergents as a buer ingredient due to their tendency to destroy lipid membranes. The outer membrane protein TtoA was immobilized to an ATR crystal in order to study the fold-ing processes of TtoA as well as the chaperone function of the insertase TtOmp85 by ATR-FTIR spectroscopy. Therefore, 3-(triethoxysilyl)propyl isocyanate was covalently bound to the silicon IRE via the proposed addition-elimination reaction of trialkoxysi-lanes with glass substrates by Schlecht and Maurer.^[197] Nα,Nα -bis(carboxymethyl)-L-lysine (also: aminonitrilotriacetic acid, or ANTA) in H2O was added to equip the linker with an NTA group. The nucleophilic amine group of ANTA reacts with the isocyanate group. This isocyanate group of 3-(triethoxysilyl)propyl isocyanate shows a band at 2267 ⁻¹ which disappears upon addition of H2O or ANTA because the isocyanate group is either replaced by substituted urea or an amine, respectively. The reaction of H2O with isocyanate competes with ANTA addition and restricts the number of NTA head groups available for protein immobilization. Although primary and secondary amines show a higher reaction rate with isocyanate than H2O, the vast excess of water molecules reduces the yield of the required amine reaction with the isocyanate group.

Thus, the concentration of ANTA should be kept high in order to optimize the yield.

The alternative use of non-aqueous solvents is precluded by the low solubility of ANTA in these solvents. Further, the open ATR cell leads to rapid evaporation of volatile solvents on the IRE which is another reason for the use of H2O as solvent. When the formation of linker conjugate is complete, NiCl2 is added to activate the NTA head group. In the following we refer to the construct, consisting of the IRE-immobilized silane-NTA derivative with Ni²⁺, as activated silane-NTA linker.

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

Figure 5.19: Formation of the IRE-linked silane-NTA conjugate. Upper panel: 3-(triethoxysilyl)propyl isocyanate can either react with ANTA (1) or with H2O (2). (SiOCH2CH2, isocyanate- , urea- and primary amine groups are marked in green, red and blue, respectively.) Lower panel: 3-(triethoxysilyl)propyl iso-cyanate absorbs at 2267 cm⁻¹ (black). Isocyanate reacts with ANTA or H2O resulting in disappearance of the absorbance band at 2267 cm⁻¹ (red). The neighboring bands are of CO2.

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

5.2.2 Immobilization of native, His-tagged protein to Ni

-activated, IRE-bound silane-NTA linker

An IRE with Ni²⁺-activated silane-NTA linker was incubated with 35 µL native, His-tagged TtoA in FC-12 buer (20 mM Tris, 100 mM NaCl, 0.1 %FC-12, pH 7.4). The crystal was then rinsed to remove unbound TtoA. The spectra recorded after rinsing show immobilized protein (Figure 5.20). The split signal at 1627 cm⁻¹, 1638 cm⁻¹ and 1687 cm⁻¹ was assigned to antiparallel β-structure, as found in the transmembrane β-barrel. The presence of two low-frequency bands, 1627 cm⁻¹ and 1638 cm⁻¹, might indicate dierent H-bondedβ-structures.^[191]The band at 1656 cm⁻¹cannot be assigned to one dened structural element. About 60 % of the 205 amino acids, as known from crystal X-ray diraction, contribute to β-sheets whereas the remaining residues form two shortα-helices, seven β-hairpins and fourteen β-turns.^[93] These elements all contribute to the TtoA absorbance spectrum but are dicult to separate. Hence, the 1656 cm⁻¹ band will be summarily assigned to secondary structure includingα-helices, β-turns and loops. The spectrum of native TtoA was time-stable. Because TtoA binds to the linker via a His-tag, it should be possible to remove it by adding imidazole at appropriate concentrations to the crystal. The experimental verication that binding of TtoA to the linker is His-tag specic by addition of imidazole is shown in Figure 5.21.

Figure 5.20: Native TtoA was incubated with activated linker (black) and subsequently ushed (red).

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

Figure 5.21: Native, His-tagged TtoA (black line) is removed from the linker by addition of 330 mM imidazole to the sample. The bands at 1634 cm⁻¹, 1624 cm⁻¹ and 1697 cm⁻¹are assigned toβ-structure. The band at 1657 cm⁻¹is assigned to heterologous structure elements. The bands disappear when imidazole is added to the crystal (red line).

In order to further verify that Ni²⁺-conjugated NTA-head groups were responsible for protein binding, IRE-bound 3-(triethoxysilyl)propyl isocyanate was incubated with H2 O only. Ni²⁺ and native, His-tagged TtoA were added to the linker (Figure 5.22).

The band positions at 1625 cm⁻¹, 1634 cm⁻¹ and 1693 cm⁻¹ dier from the ones discussed above, but the splitting of theβ-signal into one low- and two high-frequency bands remains. 1656 cm⁻¹ is assigned to α-helices, β-turns and loops. After rinsing the IRE no TtoA remains on the IRE (Figure 5.22, red spectrum). This shows that TtoA binding requires an NTA-head group.

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

Figure 5.22: Native TtoA was added to the IRE providing silane-NTA linker molecules that had only amide and no NTA head groups and thus were unable to chelate Ni²⁺

(black). After one hour, the IRE was rinsed and the TtoA spectrum disappeared (red).

In order to verify that Strep-tagged insertase does not bind to the linker, Strep-tagged, folded TtOmp85 was added to the solution on the IRE with the silane-NTA linker (Figure 5.23). The black spectrum indicates TtOmp85 secondary structure with bands at 1629 cm⁻¹ and 1640 cm⁻¹. The signal is weak due to low TtOmp85 concentration.

This leads to a low signal-to-noise ratio but the most intense bands are at 1629 cm⁻¹ and 1640 cm⁻¹, indicating the presence ofβ-sheet structure. After rinsing no TtOmp85 remained on the crystal, verifying that no unspecic protein binding to the linker takes place (Figure 5.23, red spectrum).

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

Figure 5.23: Strep-tagged TtOmp85 was added to the IRE with Ni²⁺-activated silane-NTA linker (black). After rinsing of the IRE, the TtOmp85 spectrum disappeared (red). No interaction of TtOmp85 with the linker was observed.