Molecular Trajectories Provide Signatures of Protein Clustering and Crowding at the Oil/Water Interface

Using high throughput single-molecule total internal reflection fluorescence microscopy (TIRFM), we have acquired Molecular trajectories of bovine serum albumin (BSA) and hen egg white lysozyme during protein layer formation at the silicone oil-water interface: The-Se trajectories were analyzed to determine the distribution of molecular diffusion coefficients, and for signatures of molecular crowding/caging, including subdiffusive motion and temporal anticorrelation of the instantaneous velocity vector. The evolution of these properties with aging time of the interface was compared with dynamic interfacial tension measurements. For both lysozyme and BSA, we observed an overall slowing of protein objects, the onset of both subdiffusive and anticorrelated motion (associated with crowding), and a decrease in the interfacial tension with aging time For lysozyme, all of these phenomena occurred virtually Simultaneously, consistent with a homogeneous model of layer formation that involves gradual crowding of weakly interacting proteins. For BSA, however, the slowing occurred, first, followed by the signatures of crowding/caging, followed by a decrease in interfacial tension, consistent with a heterogeneous model of layer formation involving the formation of protein clusters. The application of microrheological methods to single molecule trajectories described here provides an unprecedented level of mechanistic-interpretation of interfacial events that occurred over a wide range of interfacial protein coverage.