## 2 Results and Discussion
A possible explanation for both the observed complexity in the force spectra and the relatively low reduction in unbinding probability when blocking the GFOGER peptide may be found when  
Figure 2: (**A**) Representative force spectra for the interaction of I-domain with GFOGER (solid line) and GPP (dashed line). The spectra correspond to 190 (GFOGER) and 125 (GPP) individual F-D curves respectively; (**B**) Specificity tests illustrating the unbinding probability observed for both uninhibited (solid grey) interactions and after inhibition with free I-domain (dashed shading). Both the GFOGER and GPP experiments were repeated four times using fresh cantilevers and the reported values represent means of these experiments. considering the molecular origins of the interaction force. For a simple model ligand-receptor pair such as biotin-avidin the complementary binding site is very specific and localised. In the case of an optimal experiment, only unbinding with an individual pair should be expected, however due to the difficulties associated with these type of experiments, both multiple simultaneous and sequential unbinding events may occur (Figure 3A). Such events help to explain the occurrence of a small number of additional peaks as observed previously. The current interaction however is inherently expected to be more complex, since the previously reported adhesion assay data indicates that both the GFOGER sequence and the flanking GPP residues may interact with the I-domain [18]. Although the GPP interaction is thought to be weak, at the molecular level it is possible that multiple bonds may form with the long, triple helical strands, at various points along the ligand. Sequential unbinding of the GFOGER peptide, with these concepts in mind, is illustrated schematically in Figure 3B. An array of different configurations may be proposed including combinations of both multiple simultaneous and sequential unbinding that therefore give rise to slightly different detachment forces leading to the multi-peaked force spectra observed. Such a situation would also mean that attempts to block the ligand would be quite difficult due to the number of available binding regions and because of steric hindrance. In order to further analyse the two data sets, a multi-component analysis was performed as shown in Figure 4. A model comprising of a sum of six and nine Gaussian components was constructed for the GFOGER and GPP data sets respectively. As can be seen, both data sets are reasonably well fit to the  
Figure 3: Schematics illustrating possible interactions of tip bound ligands and substrate bound receptors as in typical SMFS experiments. (**A**) Possible interactions associated with a simple ligand-receptor pair such as biotin-avidin; (**B**) Illustration of possible interactions with the triple helical GFOGER peptide and the I-domain. The ligand is thought to interact strongly with the GFOGER hexapeptide sequence, but may also interact with the flanking GPP residues. model and therefore estimates of the component peak heights and standard deviations were obtained as summarised in Table 1. Identifying any single peak that represents specific interaction of the hexapeptide sequence alone is very difficult. It appears in fact that many of the small sub-peaks occur at very similar forces for both the GFOGER and the GPP data. The role of GPP sequences in the interaction with I-domain therefore at this loading rate plays a much more significant role than might have been expected. Another point that should be addressed is the similarity in unbinding force found between the different peptides in the current work as compared with the large difference in adhesion observed using the ELISA assay. The key point is that these measurements were conducted at a single loading rate and therefore represent a single point on a dynamic spectrum of forces [25]. Although a similar magnitude of unbinding force was observed at this specific loading rate, it is likely that the unbinding forces will diverge across the loading rate spectrum.