Typical force–distance curves recorded during such experiment are represented in Fig. 4a. These data were recorded on a sample similar to that used for the nano-mechanical

mapping, namely RC-His12-LH1-PufX molecules immobilised on EG3/Ni2+-NTA-functionalised gold surfaces, but at a much higher surface Angiogenesis inhibitor density of ~4,000 molecules per μm2 (see Fig. 4b). For comparison, a tightly packed monolayer of RC-His12-LH1-PufX complexes 12 nm in diameter would represent nearly 7,000 molecules per μm2. The particular set of force–distance curves in Fig. 4a clearly displays unbinding events with rupture lengths in the range 2–5 nm and rupture forces in the range 165–225 pN. Typically, series of around 1,000 force–distance curves were recorded over different locations of the sample under conditions that favour or disfavour the binding of the probe-bound cyt c 2 to RC-His12-LH1-PufX complexes. Each series of force–distance curves was analyzed to evaluate the distribution of the separation forces acting

between the two proteins as well as the binding frequency under different conditions (see “”Materials and methods”"). Fig. 4 Conventional force spectroscopy. a Typical force–distance curves recorded upon the retraction of the AFM probe functionalised with pre-reduced cyt c check details 2-His6 under white light illumination recorded on a gold surface densely covered with immobilised RC-His12-LH1-PufX; b AFM topography image of NU7026 cost Roflumilast a functionalised gold surface densely covered with immobilised RC-His12-LH1-PufX; c typical force–distance curves recorded with cyt c 2-His6-functionalised AFM probe under the same conditions as the data in a but on a clean EG3/Ni2+-NTA-functionalised gold surface

(no RC-His12-LH1-PufX immobilised on it); d AFM topography image of a clean EG3/Ni2+-NTA-functionalised gold surface. The scale bar for the topography images in b and d is 500 nm. For clarity the force–distance curves in a and c are offset along the Y-axis; the scale bar for the Y-axis is 100 pN In order to exclude the non-specific interactions from our force spectroscopy experimental data, we also performed a control measurement with a functionalised AFM probe (cyt c 2-His6 attached to the tip) on a bare EG3/Ni2+-NTA-functionalised gold surface with no immobilised RC-His12-LH1-PufX complexes (Fig. 4d). In order to clearly show the difference between the rupture events occurring when separating the RC-His12-LH1-PufX and cyt c 2 proteins and the non-specific interactions in our experiment, a typical set of force–distance curves recorded over the clean EG3/Ni2+-NTA-functionalised gold substrate is shown in Fig. 4c, exhibiting lower rupture forces. The histogram in Fig. 5a shows the distribution of the rupture forces measured from 261 unbinding events over 880 force–distance curves recorded under photo-oxidative conditions (white light illumination).