## 2 Results and Discussion  
A variety of force-displacement curves were observed for the interaction of the I-domain with either GFOGER or GPP as shown in Figure 1. Figure 1A shows an example of a single or multiple simultaneous unbinding event, characterised by the delayed non-linear extension profile immediately preceding rupture. The non-linear form is widely considered to be associated with the extension of the flexible PEG linkers [23, 24] and is a useful indicator of the specificity of the unbinding. In Figure 1B an example of sequential unbinding between the I-domain and GFOGER is shown as indicated by the canonical cascade of rupture events. Most interesting however is that there appears to be some specific interaction between the I-domain and GPP peptide as illustrated again by the characteristic non-linear retraction profile shown in Figure 1C. Sequential unbinding is also observed for this pair as shown in Figure 1D. Figure 2A shows two representative force spectra for the interaction of the I-domain with GFOGER (solid line) and GPP (dashed line). Both spectra are comprised of a single large broad peak spanning 0-\(100\,\mathrm{pN}\) with a series of smaller sub-peaks superimposed. The particular form of the spectra contrasts with other interactions reported in the literature, such as the model biotin-avidin pair, where predominantly only one or two large peaks are observed over similar force ranges [22, 25, 26, 27, 28, 29], including our measurements using the same instrument used here. In order to test the specificity of the two interactions blocking tests were conducted in which free I-domain was flowed into the AFM fluid cell in order to block the GFOGER and GPP peptides. For each data set four new cantilevers were used and the values given represent means. When blocking the GFOGER peptide a small (one third) reduction in unbinding probability from \(20\%\) to \(14\%\) was observed. The unbinding probability for the uninhibited interaction of GPP with the I-domain was found to be \(8\%\). Upon blocking, this decreased by three-quarters, from \(8\%\) to \(2\%\). The decrease in unbinding probability when comparing the uninhibited interaction of GFOGER with I-domain to that of the blocked interaction of GPP was \(90\%\). This strongly indicates that specific interactions were being measured since the GFOGER peptide contains GPP sequences as well, and therefore the large reduction can only be attributed to the absence of the specific  
Figure 1: Representative force-displacement curves illustrating the interaction of the I-domain with either GFOGER or GPP. **(A)** Single or multiple simultaneous unbinding of GFOGER; **(B)** Sequential unbinding of GFOGER; **(C)** Single or multiple simultaneous unbinding of GPP; **(D)** Sequential unbinding of GPP. All types of curve may be observed within an experiment. GFOGER sequence. Two things however require further explanation; firstly that specific interactions were measured at all for the GPP ligand and secondly that the reduction in unbinding probability was only \(30\%\) when blocking the GFOGER ligand. Previously it had been shown [18] when comparing various peptide sequences using enzyme-linked immunosorbent assay (ELISA) that the GFOGER peptide interacts most strongly with the I-domain. In contrast the GPP peptide, lacking any specific guest sequences, showed so little adhesion that it was often considered as a negative control. However, the absorbance and therefore adhesion observed for the GPP interaction in that work was small but not zero, and it was also greater than the interaction of I-domain with BSA (bovine serum albumin), another negative control for which there is no basis for specific interaction. Furthermore, a weak interaction between GPP and GPVI has also recently been proposed [30].