Quantifying cation-π interactions in marine adhesive proteins using single-molecule force spectroscopy

Abstract

In marine adhesives, cation-π interactions play an important role in their liquid-liquid phase separation process and underlying their strong interfacial bonding. However, it remains challenging to study the strength of cation-π interactions at the single-molecule level. Here, we engineer a recombinant chimeric polyprotein containing the mussel foot proteins-5 (MFP5) and a finger print domain GB1 to unambiguiously quantify the strength of cation-π interactions using atomic force microscopy (AFM)-based single-molecule force spectroscopy. Our results show that the formation of intermolecular cation-π interactions can be triggered at elevated salt concentrations, consistent with previous ensemble studies. Individual cation-π interaction ruptures at about 70 pN at a pulling speed of 1.6 μm s−1, comparable to the strength of other non-covalent interactions. The strength of cation-π interactions is weakly dependent on pH, which is in stark contrast with the hydrogen bonding and charge-charge interactions. Moreover, we find that the position of the cation-π bonds are formed randomly along the polyprotein chains. The propensity of forming long range cation-π interactions increases considerably when increasing the pH from 4 to 8, presumably due to the neutralization of the positive changes of MFP5. Our study directly quantifies the mechanical strength of cation-π bonds in the biological relavent settings and reveals key design parameters that may inspire the design of biomimetic strong underwater adhesives.