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

Abstract

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. These 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.

Exploring the frontiers of colloidal behaviour where polymers and particles meet

“…lysozyme) which possess low susceptibility for molecular unfolding at the interface. The heterogeneous mechanism (ii) is important for 'soft' globular proteins like bovine serum albumin and b-lactoglobulin which exhibit a pronounced tendency to unfold and associate into clusters and aggregates following protein adsorption (McUmber, Larson, Randolph, & Schwartz, 2015). Fig.…”

Kinetics and Competing Mechanisms of Antibody Aggregation via Bulk- and Surface-Mediated Pathways

“…In those cases, hereafter referred to as surface-mediated aggregation, proteins putatively unfold and aggregate because of exposure to vaporliquid, liquid-liquid, solid-liquid interfaces, or a combination of multiple interfaces (e.g., 3-phase contact line). [13][14][15][16][17][18][19] Stirring or shaking liquid formulations with an air headspace has been shown to induce the formation of larger protein particles that can be observed using turbidity and flow-imaging with visible light. 14,20 The elimination of air-liquid interfaces can greatly reduce agitation-induced aggregation in those cases.…”

Ionic hydration-induced evolution of decane–water interfacial tension

“…For example, a decrease of the dynamic interfacial tension with aging time provides signatures for protein clustering and crowding processes at the oil-water interface. 5 Because of the micro-nano scale feature of the porethroat channels in the petroleum reservoir, IFT is crucial to understand the oil-gas-water multiphase flow dynamics in porous media, 6 including the detachment of adhered oil from rock surfaces, the displacement of trapped oil and the crude oil emulsion stability, and so on.…”