The best Gaussian fit of the histogram gives two values for the most probable unbinding force, 164 ± 19 and 305 ± 25 pN (mean ± SE), respectively. Fig. 5 Specificity of the unbinding events. a Force distribution (most probable force obtained from the Gaussian fit, blue curve) for the specific unbinding between RC-His12-LH1-PufX and the cyt c 2-His6 under white light illumination; b control measurements: distribution of forces measured on chemically reduced RC-His12-LH1-PufX complex (RC[red]) in the dark; c control
measurements: blocking the Quisinostat in vitro docking site GS-1101 in vitro RC-His12-LH1-PufX with free c 2-His6 injected during the force measurements; d control measurements: histogram showing the distribution of interaction forces measured between the cyt c 2-His6-functionalised AFM probe and a clean EG3/Ni2+-NTA-functionalised gold substrate In order to test the inhibition of the formation of a transient bound state between the RC-His12-LH1-PufX and cyt c 2-His6 proteins, we performed a control experiment similar to that used find more for the PF-QNM by recording a series of force–distance curves on a RC-His12-LH1-PufX complex (immobilised
on functionalised gold substrate) chemically reduced in the dark to prevent RC photo-oxidation. Analysis of the force data recorded under these conditions revealed a dramatic drop in the binding frequency—only 101 force–distance curves out of 1,495 exhibited rupture events resulting in a binding frequency of 6.7 % with no prominent peak observable in the force distribution histogram, Fig. 5b. The docking site on the photooxidised RC-His12-LH1-PufX was blocked with pre-reduced cyt c 2-His6 molecules that were injected into the AFM liquid cell at a final concentration of 3 μM, an order of magnitude higher than the K D of ~0.3 μM (Tetreault et al. 2001). Analysis of the data obtained after the blocking with free cyt c 2-His6 revealed a weak peak at around 180 pN in the force distribution histogram with a binding frequency of 8.8 % (140 rupture events out of 1,590 force–distance curves), Fig. 5c. This residual binding probability in the blocking control is likely to arise from
repeated binding and unbinding events between the RC-His12-LH1-PufX complex on the sample surface and the free cyt c 2-His6 in solution that leave the RC binding site unblocked for short periods. Levetiracetam Thus, each cyt c 2 docking site on the surface-bound RCs is transiently available to interact with cyt c 2 on the probe, although with a much reduced probability (29 % down to 8.8 %). Finally, the distribution of the forces recorded using a clean EG3/Ni2+-NTA-functionalised gold substrate, with no RC-His12-LH1-PufX complexes (Fig. 5d) gives no prominent peak in the histogram and the data reveal a very low frequency (~6 %) for interaction, with only 60 rupture events out of 950 force–distance curves. Discussion The cyt c 2 docking site on the RC is surrounded by the extrinsic C-terminal regions of the LH1 complex.