The most probable values for the unbinding forces were obtained from the maximum of the Gaussian fit to the force distribution combined in a statistical histogram. Normally, the rupture forces of a few hundred rupture events were compiled in force or loading rate distribution histograms. Results Surface-immobilised RC-LH1-PufX protein complexes An epitaxial gold surface was functionalised with a self-assembled monolayer of a mixture of alkanethiols with polyethylene glycol (EG3) and nitrilotriacetic acid (NTA) functional end-groups. The monomeric

RC-LH1-PufX core complex was attached to the NTA-alkanethiols via a C-terminal GW786034 manufacturer His12-tag on phosphatase inhibitor the RC H-subunit. Cyt c 2 molecules, each also carrying a C-terminal His6-tag, were immobilised onto a gold-coated (on the tip side) AFM probe also functionalised with a mixed EG3/NTA thiol monolayer (Fig. 2). The His-Ni2+-NTA coordination bond has been demonstrated

to provide the appropriate orientation and high mobility when coupling biological molecules (Dupres et al. 2005; Verbelen et al. 2007). In addition, the presence of EG3 Ro-3306 nmr end-groups in the mixed monolayer minimises the non-specific adsorption/interactions between the protein complexes and the surface or AFM probe (Vanderah et al. 2004). Fig. 2 Protein complex attachment chemistry. Schematic representation of the immobilised proteins on the AFM probe and sample substrate: The RC-His12-LH1-PufX core complexes are immobilised via His12-Ni2+-NTA coordination bond on functionalised epitaxial gold substrate. The surface density of the molecules is ~250–350 molecules per μm2. The cyt c 2-His6 molecules are attached

to a functionalised gold-coated AFM probe again via His6-Ni2+-NTA coordination bond Flavopiridol (Alvocidib) at much higher surface density of around 5,000–6,000 molecules per μm2 The surface density of the immobilised RC-His12-LH1-PufX molecules on the functionalised epitaxial gold surface was found to be in the range 250–350 molecules per μm2, while the surface density of the cyt c 2-His6 molecules attached to the functionalised AFM probe was estimated to be much higher, in the range of 5,000–6,000 molecules per μm2. This is equivalent to 100–150 cyt c 2-His6 molecules for the active area of the tip (see “”Materials and methods”").