Redefining Fibrinogen Self-Assembly at the Air–Water Interface: An Intriguing Story with Multiple Layers

Abstract Layer-by-layer self-assembled structures of the blood-clotting glycoprotein fibrinogen have been resolved at a fluid interface using neutron reflectometry and complementary techniques to reveal an unexpected mechanism that contradicts a long-established adsorption model. The widely accepted monolayer tilt model, inferred over two decades ago from ellipsometry data, involves progressive tilting of a monolayer of fibrinogen molecules from parallel with the interface with increasing interfacial coverage until, at saturation, the molecules adopt a more vertical conformation. Our new structural framework is fundamentally different in that fibrinogen is shown unequivocally to orient parallel with the interface in multiple discrete layers, and, with increasing bulk concentration, there is enhanced layer thickness, greater coverage of the submerged layers and, in cases, the presence of additional layers. The varying layer thickness is attributed to changes in the conformational freedom of different monomer/dimer interactions at the interface. The driving forces for the layer-by-layer self-assembly are discussed in terms of intermolecular electrostatic interactions, hydrogen bonding and hydration, and conformational adaptability. The universal relevance of our new model is demonstrated by data from experiments performed in acidic and basic buffers over a wide range of bulk concentrations and at different ionic strengths. Our new findings have broad implications for interfacial protein behavior, as they help to explain the mechanism of sealing open wounds through scab formation at the protein-rich fluid interface of blood, the enhanced competition for interfacial area from plasma proteins over phospholipids impairing lung function in patients with acute respiratory distress syndrome, and the strong innate immune recognition in nanoparticle protein coronas.