αSNAP Binding to the Complex Monitored Calorimetrically . 52

The fluorescence experiments in section 4.1.6, investigating αSNAP-binding, indi-cated that more than 17 αSNAPs per complex were needed to achieve saturation.

This finding dramatically differs from the 3:1 ratio one would expect and might be explained either by a reduced affinity or efficiency or alternatively by degradation, which renders a large portionαSNAP incompetent with respect to SNARE-complex binding. To check, whether the recombinant αSNAP binds the SNARE complex in

a stoichiometric ratio of 3:1 when used at higher concentrations, isothermal titration calorimetry was performed. The calorimeter detects temperature changes with such high sensitivity that it can be used to quantify the small amounts of heat released or consumed during protein/protein interactions. One protein is titrated into the other in distinct steps in an adiabatic jacket, and enthalpy changes as well as the stoichiometric ratio can be directly measured.

0 , 0 0 , 5 1 , 0 1 , 5 2 , 0 2 , 5 3 , 0 3 , 5 4 , 0 4 , 5 5 , 0 5 , 5 6 , 0

- 6 - 4 - 2

0

- 0 , 1 2 - 0 , 0 8 - 0 , 0 4 0 , 0 0

- 1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

T i m e ( m i n )

µcal/sec

M o l a r R a t i o

kcal/mole of injectant

Figure 4.17: Isothermal titration calorimetry of αSNAP binding to the core complex.

αSNAP (70µM) in the syringe was titrated in ten steps into purified ternary SNARE complex (H3/SNAP25/Sb, 2,5µM) in the cell. Shown are the enthalpy changes during the experiment on top, and the integrated areas normalized to the amount of αSNAP (kcal/mol) versus the molar ratio ofαSNAP to the SNARE complex below. The solid line represents the best fit to the data for a three site sequential binding site model using a nonlinear least squares fit.

As can be seen in figure 4.17, the enthalpy changes are very low obscuring the possibilities to extract much thermodynamical information. The stoichiometry ’n’

of the SNARE complex could nevertheless be determined to be 3 αSNAPs per complex in these experiments. Notably, this is exactly what is expected and hence allows us to conclude, that the majority of the recombinant αSNAP is able to bind SNARE complex, excluding the possibility that degradation is the reason for the low potency. Notwithstanding the low enthalpy values of the reaction, I still tried to fit the reaction in order to roughly estimate the αSNAP affinity. The thermodynamic data could not be properly fitted using a single site binding model, which assumes thatαSNAP has the same affinity to all SNARE binding sites. Using a three-site sequential fit however, the curve could nicely be fitted (see figure 4.17).

This suggests, that αSNAP binding to SNARE complexes is not equal to all three SNAP/SNARE interaction sites but the sites rather display different affinities. The affinities resulting from this fit were 10nM for the first, 50nM for the second and 380nM for the third binding site. Nevertheless, keeping in mind the low enthalpies and also considering that only a low number of data points per binding site were recorded, the affinities of αSNAP to the SNARE complex determined might not be very reliable.

4.3.2 αSNAP Dependence of SNARE Disassembly on ’Mem-brane Sheets’

Having established thatαSNAP binds the SNARE complex at the expected ratio of 3:1, two possibilities remain: Either does the recombinant αSNAP have a reduced affinity as opposed to the endogenous protein, or a factor important for binding is missing in our fluorescence assay. Hence, the next step was to test the recombinant αSNAP’s efficacy under the more physiological conditions of a different system, the so called membrane sheets. These membrane sheet experiments experiments were conducted together with Dana Bar-On.

Membrane sheets are generated by subjecting cultured, adherent PC12 cells to a gentle ultrapulse. The upper half of the cells is consequently ripped of, leaving behind an intact native membrane bilayer attached to a cover slip. The preparation provides the full set of membrane lipids and proteins in their native environment, including steric and conformational restriction, membrane fluidity, cytoskeletal com-ponents, lipid component diversity, and associated regulatory factors.

Figure 4.18: Disassembly of SNARE complexes on membrane sheets. After completion of complex formation, the membranes were incubated with increasing concentrations of NSF andαSNAP in the presence of Mg²⁺/ATP, allowing for disassembly of the formed SNARE complexes. Upon disassembly the soluble fluorescent synaptobrevin is released into the buffer making the observed loss of fluorescence from membrane sheets a direct read-out of the disassembly reaction (100% represent total fluorescence before disassembly).

Cis-SNARE complexes in this experiment were generated by pre-incubation of membrane sheets with the fluorescent recombinant Synaptobrevin2 also used in the fluorescence assays. As in solution, the Synaptobrevin spontaneously assembles with its cognate t-SNAREs, here the endogenous Syntaxin and SNAP25 sitting on the PC12-cell membrane. After completion of complex formation, the membranes were incubated with increasing concentrations of NSF andαSNAP in the presence of Mg²⁺/ATP(5mM), allowing for disassembly of the formed SNARE complexes.

Upon disassembly, the soluble fluorescent synaptobrevin is released into the buffer making the observed loss of fluorescence from membrane sheets a direct read-out of the disassembly reaction. As illustrated in figure 4.18, fluorescence decreases with time in an NSF- and αSNAP-concentration dependent manner.

Surprisingly, under the conditions of the membrane sheet assay ∼100nM of αSNAP are sufficient to mediate disassembly equally well as 2µM indicating sat-uration at 100nM. Of course the amount of SNARE target in these assays is not

known, not permitting a conclusion about the stoichiometry ofαSNAP and SNARE complexes in this assay. Nevertheless, together with the ITC measurements which indicated a molar ratio of 3:1, these findings strongly suggest that a factor miss-ing in the FRET and anisotropy experiments but present on membrane sheets is responsible for the higher αSNAP affinity.

4.3.3 Disassembly of SNARE Complexes Incorporated into