Kinetics and Structure of Self-Assembled Flagellin Monolayers on Hydrophobic Surfaces in the Presence of Hofmeister Salts: Experimental Measurement of the Protein Interfacial Tension at the Nanometer Scale

Abstract

In the present study, we monitor the adsorption–desorption kinetics and adsorbed layer structure of the bacterial protein flagellin in the presence of Hofmeister salts by a surface sensitive label-free optical biosensor (optical waveguide lightmode spectroscopy, OWLS). The recorded OWLS data were analyzed by a computer code using a set of coupled differential equations modeling the adsorption–desorption process. By supposing reversibly and irreversibly adsorbed protein states with different adsorption footprints, the kinetic data could be perfectly fitted. We revealed that the proteins adsorbing in the presence of kosmotropic salts had smaller footprints, leading to a more oriented and densely packed layer. Kosmotropic salts increased both the adsorption rate constant and the transition rate constants from the reversibly to the irreversibly adsorbed state. In contrast, chaotropic salts increased the desorption rate constant and led to decreased adsorbed mass and a more loosely packed film. Neither circular dichroism spectroscopy in bulk solutions or Fourier transform infrared spectroscopy of surface-adsorbed flagellins could reveal significant structural changes due to the presence of the Hofmeister salts, and supported our conclusions about the adsorption mechanism. On the basis of the measured kinetic and structural data (footprints of adsorbed proteins), we developed a model to calculate the protein–water-substrate interfacial tension in the presence of Hofmeister salts, and compared the experimentally obtained values with related literature data. The calculated values are consistent with previously published data of surface tension changes, and—to the best of our knowledge—represent the first experimental results for this quantity.