The Influence of Cross-Linking on Protein-Protein Interactions in a Marine Adhesive: The Case of Two Byssus Plaque Proteins from the Blue Mussel

The interaction between two proteins, Mefp-1 and Mefp-2, from the byssal plaque of the blue mussel, Mytilus edulis, was investigated using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. The challenge in using a surface-sensitive technique to investigate the interaction between two strongly adhesive proteins was met by coupling a biotinylated version of one of the proteins (b-Mefp-1) to an inert two-dimensional arrangement of streptavidin (SA) formed on top of a biotin-doped supported phospholipid bilayer. The interaction between Mefp-1 and Mefp-2 was investigated by addition of Mefp-2 to SA-coupled b-Mefp-1, where the latter was either in the native state or cross-linked using sodium periodate (NaIO4), Cu2+ or mushroom tyrosinase. Using this coupling strategy it is shown that a requirement for association between the two proteins is that tyrosinase is used as the cross-linking agent of b-Mefp-1 coupled to SA. By inhibiting the enzymatic activity of tyrosinase it is also shown that enzymatic activity is required for both efficient binding of tyrosinase to SA-coupled b-Mefp 1 as well as for the subsequent binding of Mefp 2. In contrast, spontaneous adsorption of Mefp-1 to a methyl-terminated (thiolated) gold surface followed by addition of Mefp-2 results in binding of Mefp-2 for all cross-linking agents. This suggests that cross-linking of Mefp-1 adsorbed on a solid surface induces structural changes in the adsorbed protein layer, resulting in exposure of free surface patches on which Mefp-2 binds.