Proteins at soft interfacesa self-assembly, structure, kinetics and function study

Resumen

The PhD thesis reports the adsorption and immobilization of two dissimilar proteins, bacterial SbpA and Human Serum Albumin, on soft surfaces. These proteins have been selected due to differences in structure and biological function. The thesis shows different strategies for surface chemistry modification that permit to generate crystalline surface layers, maintaining constant the lattice parameters, the thickness, the adsorbed protein mass, but changing the protein crystalline domains size and the protein adsorption rate. Through the combination of Atomic Force Microscopy (AFM) and Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), the mechanism of the S-layer formation has been clarified at nanoscale resolution for the first time. First, S-protein adsorbs on surface forming nucleation points; second, S-proteins start the self-assembly process that build the protein crystal layer (S-layer); and third, the protein crystal domains reorganize themselves to minimize the amount of surface defects and therefore the surface energy. Furthermore, the thermal stability of Human Serum Albuminm (HSA) and the free energy and binding constant of HSA-ibuprofen and HSA-bilirubin complexes have been determined. In addition, systematic immobilization studies permitted to find the best substrate for HSA adsorption at physiological pH. HSA immobilization and a new strategy to functionalize tips allow to investigate the interaction between ibuprofen and HSA, obtaining a first description of the energetic landscape: the distance of the transition state and the dissociation rate constant at zero force. These results might indicate that albumin loses functionality through surface immobilization.