S-layer recrystallization through hydrophobic/hydrophilic nanoprotrusions

Bacterial surface layer proteins (S-layers) have the ability to build protein crystal layers with nanometer regularity on solution and many different substrates. They are currently being tested as nano-templates for different biotechnological applications. However, the (path)way in which such proteins self-assemble forming organized nanostructures is not fully understood. In this context, we propose to investigate the recrystallization of three S-layer proteins, wild type SbpA and the recombinant proteins rSbpA311068 and rSbpA31-918, on (molecularly controlled) hydrophobic and hydrophilic disulfides. First, we will study the adsorption kinetics and recrystallization of the three bacterial proteins. Second, we would like to find the relation between the kinetics and the physical properties of the formed protein crystal (e.g. crystal domain size, lattice parameters). Third, we would like to clarify the question of the recrystallization pathway as a function of the properties of the substrate for these bacterial proteins (which also imply to get insight about protein/substrate interactions, especially about the recognition by the protein of hydrophobic and/or hydrophilic moieties). Hypotheses The main hypotheses addressed in this project are: i) The length of the hydrophobic/hydrophilic nanoprotrusions should influence the recrystallization pathway and the adsorption kinetics; ii) Hydrophobic/hydrophilic nanoprotrusions might induce a transition from monolayer to bilayer and also different protein orientation (the three proteins should behave differently), iii) Elucidating the screening of the hydrophobic interaction by exposing hydrophilic groups to the protein, and iv) Similar proteins should follow the same recrystallization pathway (either classical or non-classical). Methods Atomic force microscopy, quartz microbalance with dissipation, electron microscopy, electrophoretic mobility, infrared spectroscopy, surface chemistry modification, cell culture and molecular biology techniques (e.g. recombinant proteins). Novelty and originality of the project The project offers a systematic study of the building of biomimetic surfaces made of bacterial proteins with different physical and chemical properties. This can be achieved by changing the length (a few C-C bonds) of the hydrophobic part of the disulfide. From the academic perspective, the proposed system enables to test the validity of classical and non-classical crystallization theories, and provide information about the interaction between the bacterial proteins and hydrophobic and hydrophilic interfaces. The control of the kinetics and the final protein crystal structure (as a new bottom-up approach) will be important for S-layer biotechnological applications (e.g. biosensing, antifouling and smart surfaces, biomineralization, etc.).

The main objective of this project was to elucidate the recrystallization pathways of three S-layer proteins. This was achieved by controlled substrate chemical modifications. Other goals included: i) the quantification of the recrystallization kinetics, the lattice parameters of the formed protein crystal layer and the crystal domain size; ii) Influence of the substrate nature and protein type on thermodynamic, kinetic parameters, and final nanostructure of the crystal protein layer, and iii) comparison of our results with classical and non-classical recrystallization theories and models. We first exposed SbpA bacterial proteins to hydroxyl-terminated (-OH) and methyl-terminated (-CH3) chemical groups. We studied protein adsorption and crystal formation with atomic force microscopy (AFM). With quartz crystal microbalance with dissipation (QCM-D) the kinetics of the process (and the amount of adsorbed protein) was quantified. Adsorption was faster on hydrophobic surfaces. The interplay protein concentration vs. measuring time for crystal formation was also studied on hydrophobic fluoride functionalized SiO2 surfaces. The results indicated: (1) crystal formation took place at concentrations above 0.08 M, (2) the crystal compliance decreased by increasing protein concentration, and (3) protein-substrate interactions seemed to prevail over protein-protein interactions. All the crystal domains observed had similar lattice parameters (a = 14.8 0.5 nm, b = 14.7 0.5 nm, = 90 2). Protein film formation started from initial nucleation points which originated a gradual and fast extension of the crystalline domains. Crystal growth could be modeled with the Avrami equation. Furthermore, dynamic-ow experiments the formation of a closed and crystalline protein lm even at low protein concentrations (i.e., 10 g/mL). This is an important result since such a protein layer cannot be formed under static flow conditions. A new probabilistic model to explain and predict 2D protein crystal growth at the microscale has been developed. This could be achieved by simulating the spatial growth of the bulk crystal without considering the individual constituents of the crystal: their orientation, the nucleation process, and/or other energetic considerations. We considered a probabilistic model and dene all the parameters involved in it. Such a model took into account the available space for growing. Finally, we have explored the antifouling properties of 2-D bacterial protein crystals for cell-surface and cell-cell interaction measurements.