5. Quantification of the Phosphoinositide-Recognition Reaction of PROPPINs 82
5.2.4. Affinity Measurement
Figure 5.3.:Measurement of adsorption isotherm of GST-ScHsv2 on a lipid membrane func-tionalised with 3 mol% of PtdIns3P. Baseline was recorded in buffer pH 6.5. At point ’a’ vesicle solution was introduced. After bilayer formation was complete, the system was washed with buffer of pH 7.0 (point ’b’). The protein was added step-wise increasing its concentration (steps given in the graph inµM) and eventually the whole system was washed with buffer (point ’c’) [172].
The measurement of absorption isotherms was commenced by recording a baseline in buffer containing 10 mM HEPES and 150 mM NaCl at pH 6.5 for at least 5 min.
Afterwards, the SUV solution was allowed to circulate over the transducer chip for several minutes. Once membrane formation was complete, the flow cell was rinsed with pH 7.0 buffer for at least 5 min. Afterwards, GST-ScHsv2 or one of its mutant variants was added stepwise (30 nM - 1.5 µM) increasing the protein concentration in each step.
Eventually the whole system was rinsed with buffer (point ’c’).
5.3. Results and Discussion
To measure the binding affinity of GST-ScHsv2 and its mutants to a PtdIns3P function-alised membrane, we first had to overcome the difficulty of preparing such a functionfunction-alised solid-supported membrane on a silicon transducer chip in a defect-free fashion. The pre-ferred procedure to form a lipid bilayer is spreading of vesicles, since it allows for a rapid membrane preparation without interfering factors such as organic solvents [53,54]. When dealing with lipid vesicles containing PtdInsP in ratios higher than 1 mol%, it is almost impossible to achieve close to 100 % surface coverage in SSM preparation, due to electro-static repulsion of the negatively charged surface and vesicles. Since we needed at least 3 mol% of PtdIns3P in the membrane to adsorb a monolayer of protein on the surface, we adjusted the pH of the buffer to a slightly more acidic value of 6.5 to circumvent this problem.
An exemplary measurement can be seen in figure 5.3. The baseline was also recorded in the more acidic buffer. The formation of a membrane can be inferred from the peak in the curve, which results from the vesicles first adsorbing on the surface followed by rupture and fusion leading to the formation of a continuous planar bilayer. To set ideal conditions for the protein and to get rid of excess vesicles, the system was washed with pH 7.5 buffer (point ’b’). As can be seen in the graph, theOT decreases again since the electrostatic repulsion is no longer shielded. If the signal fell again below 6 nm during rinsing, the surface coverage was too low, meaning parts of the transducer chip were not covered with lipid membrane. This would have resulted in a false positive experiment, since the protein was prone to adsorb on the chip surface itself. The measurement was started anew should that occur. The integrity of the lipid membrane was verified through fluorescence microscopy. The gradual addition of GST-ScHsv2 led to distinct rises inOT.
Figure 5.4.:Adsorption isotherms of GST-ScHsv2 binding to lipid membrane functionalised with 3 mol% of PtdIns3P. A: Pilot experiment measured over large concentration range. The curve shows the characteristics of a BET isotherm most likely due to GST-GST interaction. Langmuir fit of the lower concentration range indicating monolayer formation is shown in black. B: Isotherms from three different mea-surements (red, blue, green) and Langmuir fit (black line) leading to a KD value of (1.3± 0.2)µM.
The obtained adsorption isotherms for wildtype GST-ScHsv2 can be seen in figure 5.4.
The graph in figure 5.4 A displays an isotherm measured over a large concentration range (up to 5 µM). It shows the characteristics of a BET isotherm, meaning a second layer of protein might be adsorbed on top of the first one. Since the multi-layer adsorption was most likely induced by GST dimerisation rather than a characteristic of ScHsv2 itself, the following measurements were limited to the lower concentration range for which monolayer formation may be assumed (up to 1.6 µM). The three measurements in figure 5.4 B display the characteristics of a Langmuir adsorption isotherm. A dissociation constant (KD) of (1.3±0.2)µM for the GST-ScHsv2-PtdIns3P complex was determined by fitting the parameters of the Langmuir equation (chapter 3.3 equation (3.42)) to the data (fit shown in black).
5 Quantification of Phosphoinositide-Recognition of PROPPINs
The dissociation constant was also measured with isothermal titration calorimetry (ITC) by the group of Karin Kühnel. They tritated GST-ScHsv2 into liposomes consist-ing of 2 mol% PtdIns3P, 73 mol% phosphatidylcholine, 23 mol% phosphatidylethanolamine and 2 mol% Texas Red-phosphatidylethanolamine. AKD value of (0.67± 0.04)µM was found [172]. Independent from our study, Baskaran et al. determined the affinity of GST-ScHsv2 by a fluorescence resonance energy transfer (FRET) flotation assay [174].
From their published data, a KD value of about 0.4 µM may be inferred. The lowerKD values given by these two methods were expected, as we mentioned discussing the biotin streptavidin interaction, it is known that there is a loss of translational degree of freedom for interactions measured on surfaces compared to flotation assays resulting in the free energy of adsorption to be lowered by about 1.5 kBT per degree of freedom [139]. Calcu-lating the free energy of adsorption from the threeKD values, we derive 13.5 kBT for the RIfS measurement 14.3 kBT for the ITC experiment and 14.7 kBT from the FRET assay.
The values reflect very well the aforementioned relation, even though, the measurements were carried out with vesicles which on the molecular level can be regarded as surfaces as well. Since the ITC and FRET investigation are both flotation assays, the interaction of both binding partners is facilitated through a better mixing, as opposed to our surface based assay, which also contributes to the higherKD value we found. Having determined the affinity of wildtype GST-ScHsv2 to PtdIns3P, we investigated how the interaction was affected by mutations in the binding pockets of ScHsv2.
Figure 5.5.:Measurements of GST-ScHsv2 mutants. The double mutant ScHsv2-R264A-R265A as well as the mutants ScHsv2-R264A and ScHsv2-S243A for binding site 1 and ScHsv2-R265A and ScHsv2-H294A for site 2. After bilayer formation, each mutant protein was added in increasing concentrations (up to 5µM) with waiting periods of 5 min between additions. The mutant proteins showed no significant membrane interaction
Mutant variants of ScHsv2 were cultivated to probe the capabilities of sites 1 and 2 for phosphoinositide binding. To this purpose, 10 basic and polar residues in proximity to the two binding sites were selected and mutated to alanines. We measured adsorption
isotherms of the double mutant R264A-R265A as well as the mutants ScHsv2-R264A and ScHsv2-S243A for binding site 1 and ScHsv2-R265A and ScHsv2-H294A for site 2. The results can be seen in figure 5.5. The measurements with ScHsv2 mutants were carried out in an analogous manner to the wildtype measurements. We waited at least 5 min between successive additions of protein. As can be seen in figure 5.5, the mutants showed no significant interaction in the same concentration range as for the wild-type GST-ScHsv2. Higher concentrations could not be investigated due to protein aggregation. These findings led us to the conclusion that both binding sites are needed for effective membrane association of ScHsv2. The conclusion was further strengthened by the fact that a molar ratio of 0.50 ± 0.06 for protein binding to PtdIns3P was found in the ITC measurements, which corresponds very well to two PtdIns3P binding sites per PROPPIN molecule. Unfortunately, the mutant measurements were not repeated with that method.
When dealing with the determination of binding affinity, control experiments to check for non-specific interactions are needed. GST-ScHsv2 was tested against non-functionalised lipid membranes and GST itself against functionalised bilayers, but no interaction was found in both cases.
5.4. Conclusion
In conclusion we were able to provide an equilibrium dissociation constant of (1.3 ± 0.2) µM for wildtype GST-ScHsv2 bound to PtdIns3P. Furthermore, we found a loss in binding affinity for its mutants with our biomembrane assay, proving that indeed both suggested PtdIns3P binding sites are necessary for the successful membrane association of ScHsv2.