Quantification of the STxB Gb 3 interaction

To investigate the reason for the different capability of Gb₃-C24:1 2(S)-OH and Gb₃-C24:1 2(R)-OH to form membrane invaginations, the interaction of STxB with both Gb₃ derivatives was quantified using surface plasmon resonance (SPR) spectroscopy and reflectometric interference spectroscopy (RIfS).

Surface plasmon resonance spectroscopy

SPR allows to determine the change in reflectivity ∆R, which is proportional to the newly deployed material on top of the sensor surface (Section 3.3.4.1) enabling the determination of the affinity of a solute to the surface. The dissociation constantK_D was determined for two membranes systems; (i) 5 mol% Gb₃ in a 95 mol% DOPC matrix and (ii) a system composed of DOPC/Chol/Gb₃ 75:20:5. The time course of ∆R of a typical experiment is shown in Figure 4.30A. Experimental procedures were adapted from Nakajima et al..¹⁰⁷

Upon addition of vesicles in PBS (a, DOPC/Gb₃-C24:1 2(R)-OH 95:5, final concentration 0.03 mg mL⁻¹) to an octanethiol functionalized gold sensor chip the reflectivity increases indicating the formation of a hybrid bilayer. Rinsing with PBS (b) results in a slow and steady drop in reflectivity caused by the detachment of vesicles and membrane multistacks. The process is accelerated by the repeated addition of NaOH solution (c, 50 mm). Possible hydrophobic defects are blocked by bovine serum albumin (BSA, d, 1 mg mL⁻¹). In most experiments the increase in reflectivity upon BSA addition was fully reversible indicating that no hydrophobic defects were present. STxB in increasing concentrations between 1 and 120 nm was added (e to h) and the change in reflectivity compared to baseline level after BSA addition was determined. Overall absolute changes in reflectivity for membrane formation and protein binding were found to vary strongly among the

experiments probably due to differences in the roughness of the gold layer.¹⁸⁴ All reflectivity changes were normalized by dividing the value by the reflectivity change at saturation (c= 120 nm). Figure 4.30B/C show the adsorption isotherms for DOPC/Gb₃-C24:1 2(S)-OH 95:5 and DOPC/Gb₃-C24:1 2(R)-OH 95:5 together with a fit according to the Langmuir model (Equation 3.14). Fitting the data yields K_D= 9±2 nm for Gb₃-C24:1 2(S)-OH and K_D= 8±2 nm for Gb₃-C24:1 2(R)-OH containing membranes. These values are in excellent agreement with the affinity determined for the diastereomeric mixture Gb₃-C24:1 2(R/S)-OH (Table 4.1).

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Figure 4.30:SPR analysis of the binding of STxB to hybrid membranes containing 5 mol%

of Gb₃-C24:1 2(R)-OH and 95 mol% DOPC.ACharacteristic course of an experiment with a zoom in shown in B. (a): Addition of small vesicles (DOPC/Gb₃-C24:1 2(S)-OH 95:5, final concentration 0.03 mg mL⁻¹) at a flowrate of 50 µL min⁻¹. (b): rinsing. (c): flowrate was lowered to 25 µL min⁻¹ to add NaOH solution (4×50 mm) for 2 min each to remove vesicles and lipid multistacks. (d): Addition of BSA solution (1 mg mL⁻¹) to block defects in the membrane followed by rinsing with PBS. (e)-(h): Additions of STxB (c= 10, 15, 20, 120 nm) followed by rinsing with PBS.CAdsorption isotherm of STxB to membranes containing Gb₃-C24:1 2(S)-OH. Crosses denote data points and the line the fit according to the Langmuir model.D Langmuir adsorption isotherm of STxB to membranes containing Gb₃-C24:1 2(R)-OH. Crosses denote data points and the line the fit according to the Langmuir model. Image modified from Schütte et al.2015.⁸⁷

Cholesterol was added to the lipid mixture to resemble the free standing mem-brane of the GUVs more closely. A typical time course of an experiment in a DOPC/Chol/Gb₃-C24:1 2(R)-OH 75:20:5 membrane is shown in Figure 4.31A. It closely resembles the cholesterol free case. No apparent differences in membrane formation, multilayer detachment and protein binding were found.

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Figure 4.31: SPR analysis of the binding of STxB to hybrid membranes containing 5 mol% of Gb₃-C24:1 2(R)-OH, 20 mol% cholesterol and 75 mol% DOPC.ACharacteristic course of an experiment with a zoom in shown in B. (a): Addition of small vesicles (DOPC/Chol/Gb₃-C24:1 2(S)-OH 75:5, final concentration 0.03 mg mL⁻¹). (b): rinsing.

(c): Addition of NaOH solution (7× 50 mm) for 2 min each to remove vesicles and lipid multistacks. (d): Addition of BSA solution (1 mg mL⁻¹) to block defects in the membrane followed by rinsing with PBS. f: rinsing. (e)-(i): Additions of STxB (c= 1, 7, 14, 45, 120 nm) followed by rinsing with PBS. C Adsorption isotherm of STxB to membranes containing Gb₃-C24:1 2(S)-OH. Crosses denote data points and the line the fit according to the Langmuir model.D Adsorption isotherm of STxB to membranes containing Gb₃-C24:1 2(R)-OH. Crosses denote data points and the line the fit according to the Langmuir model. Image partially modified from Schütte et al. 2015.⁸⁷

The adsorption isotherms fitted according to the Langmuir model (Figure 4.31B/C) for membranes composed of DOPC/Chol/Gb₃-C24:1 2(S)-OH 75:20:5

(K_D= 13±3 nm) and DOPC/Chol/Gb₃-C24:1 2(R)-OH 75:20:5 (K_D= 14±3 nm) give twofold reduced affinity constants (Table 4.31). SPR spectroscopy provided identical affinities of STxB to Gb₃-C24:1 2(R)-OH and Gb₃-C24:1 2(S)-OH. The differences in the formation of invaginations are not caused by the protein’s affinity to the membrane. However, due to the variation in absolute changes of the reflectivity between the experiments no information on the binding capacities of the Gb₃s could be extracted.

Reflectometric interference spectroscopy

Reflectometric interference spectroscopy (RIfS) was used to determine the absolute optical thickness (OT) of the membrane and the adsorbed protein layer (Section 3.3.4.2).¹¹¹ In contrast to the SPR experiments membranes prepared for RIfS setup are bilayers formed on hydrophilic silicon dioxide wafers. Using the affinity constants obtained by SPR, the concentration of STxB used for each experiment was adjusted to give 90 % receptor occupancy. Under these conditions, it is possible to compare the binding capacities of the Gb₃ derivatives derived from the absolute changes in height (Figure 4.32). These changes are presumably also a direct measure for the valency of the STxB-Gb₃ interaction.

Gb3-C24:1 2-OH Cholesterol DOPC STxB

Averaged Δh

Figure 4.32: Schematic drawing demonstrating that the height difference measured in RIfS (∆h) is proportional to the surface coverage and presumably also indicates the valency of the STxB-Gb₃ interaction.

Typical experiments using DOPC/Gb₃ 95:5 and DOPC/Chol/Gb₃ 75:20:5 membranes are shown in Figure 4.33A/B. Addition of vesicles in PBS results in a rapid increase in optical thickness indicating the formation of a lipid bilayer. Using cholesterol containing membranes, in all experiments a slight overshoot was found, which quickly leveled off to give a constant optical thickness. Rinsing with PBS (b), addition of BSA solution (c, 1 mg mL⁻¹) and rinsing (d) did not change the

optical thickness indicating that after spreading a defect free membrane is formed.

Experiments were performed three times per lipid mixture.

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Figure 4.33: A, B Examples of typical experiments for DOPC/Gb₃ 95:5 membranes (A) and DOPC/Chol/Gb₃ 75:20:5 membranes (B). (a): Addition of small vesicles in PBS (final concentration 0.1 mg mL⁻¹, DOPC/Gb₃-C24:1 2(S)-OH 95:5 (A) or DOPC/Chol/Gb₃-C24:1 2(S)-OH 75:20:5 (B)) followed by rinsing with PBS (b). (c):

Addition of BSA solution (final concentration 1 mg mL⁻¹) to block defects in the membrane followed by rinsing with PBS (d). (e): Addition of STxB (final concentration 72 nm (A) or 117 nm (B)) followed by rinsing with PBS (f). C Bilayer thicknesses of the different systems.DThicknesses of the STxB layers after binding to the membrane. Image partially modified from Schütte et al. 2015.⁸⁷

The membrane thickness was calculated using a refractive index of 1.49¹¹² and is ∆h= 4.16±0.16 nm for DOPC/Gb₃ 95:5 membranes in good agreement with literature (Figure 4.33C).¹⁸⁵ Cholesterol increases the thickness of the membranes by 0.5 nm.¹⁸⁶

An increase in optical thickness for the binding of STxB was converted to the change in height using a refractive index of 1.47 for a 35 kDa protein (Figure 4.33D).¹¹³ The changes are summarized in Table 4.12 and lie between

∆h= 0.54±0.08 nm for DOPC/Gb₃-C24:1 2(R)-OH 95:5 and ∆h= 0.93±0.09 nm for DOPC/Chol/Gb₃-C24:1 2(S)-OH 75:20:5.

An increase in the binding capacities for Gb₃-C24:1 2(S)-OH membranes was about 30 % compared to Gb₃-C24:1 2(R)-OH both in presence and absence of cholesterol.

According to the crystal structure of STxB the protein has a height of 2.0 nm.⁶⁰ Using this value, the measured heights upon binding can be converted to surface coverages of 27 to 47 % (Table 4.12).

After rinsing with PBS, ≈70 % of the protein remained irreversibly bound to the membrane. Cholesterol increases the binding capacity of the membranes suggesting an influence on the conformation of the headgroup of Gb₃ or an alteration of the lateral organization of the receptor in the membrane.¹⁰⁷

Table 4.12: Dissociation constants K_D from SPR experiments. Thicknesses of the membrane and protein layer as determined by RIfS. Membranes composed of DOPC/Gb₃ 95:5 or DOPC/Chol/Gb₃ 75:20:5.

Gb3-C24:1 KD/nm ∆h(Mem)/nm ∆h(STxB)/nm irreversible fraction coverage

2(S)OH 9±2 4.16±0.09 0.70±0.01 74 % 35 %

2(R)OH 8±2 4.16±0.16 0.54±0.08 63 % 27 %

2(S)OH+Chol 13±3 4.71±0.24 0.93±0.09 69 % 47 %

2(R)OH+Chol 14±3 4.75±0.28 0.70±0.02 63 % 35 %

The results show a clear difference in binding of STxB and the propensity of the Gb₃ derivatives to facilitate the formation of membrane invaginations. This effect can either be an intrinsic property of the Gb₃ molecule, or be caused by the lateral organization of the receptor in the membrane.