Surface Dependence of Protein Nanocrystal Formation

The self‐assembly kinetics and nanocrystal formation of the bacterial surface‐layer‐protein SbpA are studied with a combination of quartz crystal microbalance with dissipation monitoring (QCM‐D) and atomic force microscopy (AFM). Silane coupling agents, aminopropyltriethoxysilane (APTS) and octadecyltrichlorosilane (OTS), are used to vary the protein–surface interaction in order to induce new recrystallization pathways. The results show that the final S‐layer crystal lattice parameters (a = b = 14 nm, γ = 90°), the layer thickness (15 nm), and the adsorbed mass density (1700 ng cm^?2) are independent of the surface chemistry. Nevertheless, the adsorption rate is five times faster on APTS and OTS than on SiO_2, strongly affecting protein nucleation and growth. As a consequence, protein crystalline domains of 0.02 µm² for APTS and 0.05 µm² for OTS are formed, while for silicon dioxide the protein domains have a typical size of about 32 µm². In addition, more‐rigid crystalline protein layers are formed on hydrophobic substrates. In situ AFM experiments reveal three different kinetic steps: adsorption, self‐assembly, and crystalline‐domain reorganization. These steps are corroborated by frequency–dissipation curves. Finally, it is shown that protein adsorption is a diffusion‐driven process. Experiments at different protein concentrations demonstrate that protein adsorption saturates at 0.05 mg mL^?1 on silane‐coated substrates and at 0.07 mg mL^?1 on hydrophilic silicon dioxide.