Figure 2: The tubulin-DARPin interaction monitored by fluorescence spectroscopy and surface plasmon resonance (SPR).
(a) Fluorescence variation of 100 nM (left) or 15 nM (right) acrylodan-labeled DARPins as a function of tubulin concentration. The curve is the fit of the experimental points with Equation 1, from which the KD is extracted. Error bars correspond to standard deviation from duplicate experiments. a.u., arbitrary units. (b) Dissociation of acrylodan-labeled DARPin from tubulin. In the case of D1 (left), 5.5 μM unlabeled D1 was added to a 100 nM labeled D1 and 0.5 μM tubulin mixture. The decrease in fluorescence signal was monitored in a stopped-flow apparatus (30% of the data points are shown). In the case of A-C2 (right), 2 μM unlabeled A-C2 was added to a 20 nM labeled A-C2 and 40 nM tubulin mixture and the fluorescence signal was monitored in a spectrofluorometer. The curve is the fit of the experimental points with a mono-exponential decay equation (Equation 2). (c ) Determination of the association rate constant by fluorescence. Tubulin at the indicated concentrations was added to a fixed concentration (50 nM) of labeled D1 (left) or A-C2 (right) in a stopped-flow apparatus (20% of the experimental points are displayed). The data were fitted according to Equation 3. The variation of kobs as a function of tubulin concentration is shown in inset, from which the kon (slope of the curve) is derived. (d) The tubulin-DARPin interaction monitored by SPR. D1 (left) and TM-3 (right) were immobilized through their His-tag on the sensor chip. Tubulin at the indicated concentration was applied at time zero for 60 s (D1) or 180 s (TM-3), followed by a washing buffer flow. The black curves are the fit of the experimental data using the Langmuir analysis, from which the kon and koff are extracted. In the case of D1, plotting the values at the plateau as a function of the tubulin concentration provided an estimate of the KD (Supplementary Fig. 5) that is very similar to the koff/kon ratio.
