To elucidate the molecular basis of the conjugation process, binding events were reconstructed with Molecular Dynamics (MD) simulations. To characterise the spontaneous binding process, all [60]fullerene species were initially placed in the solvent at least 10 Å away from the protein surface and were allowed to freely diffuse from the solvent to the protein surface along the MD trajectory. From these spontaneous binding MD simulations, the interactions established between the fullerene species and CTPR8 units were monitored along time to identify the most visited binding sites (hot spots) on the protein surface. In all cases, [60]fullerene species rapidly collapsed over the protein surface, establishing transient interactions with the side chains of superficial protein residues. The initial recognition with CTPR8 took place in few nanoseconds of simulation time, and the fullerene species were then displaced over the protein surface until they identified a binding site (Fig. ), where commonly they remained for the rest of the simulation. Both C 60 and C 60 A prefer to interact with the CTPR modules located at both extremes of the CTPR8 unit, while permanent interactions with the inner hydrophobic cavity were observed less frequently. To identify the most visited binding sites, representative snapshots from the last 100 ns of each replica of the MD simulations were extracted and the position of each fullerene with respect to the protein surface was plotted (colour spheres in Fig. ). CTPR8-C 60 , CTPR8-C 60 A and CTPR8Y 16 -C 60 assemblies showed a wide dispersion of fullerene molecules all over the protein surface. These results indicate that although the fullerene species tend to be in contact with protein residues, these interactions are not stable along the MD simulation and among replicas. On the contrary, for the CTPR8Y 16 -C 60 A co-assembly, fullerene molecules accumulate in selected hot spots independently of which MD simulation is analysed. These results indicate that the new tyrosine residues favour the association and stabilisation of C 60 A at specific binding sites on the protein surface. These observations are in line with the highest fullerene/protein ratio experimentally observed for the CTPR8Y 16 -C 60 A co-assembly.