Making novel bio-interfaces through bacterial protein recrystallization on biocompatible polylactide derivative films.

Fabrication of novel bio-supramolecular structures was achieved by recrystallizing the bacterial surface protein SbpA on amorphous and semicrystalline polylactide derivatives. Differential scanning calorimetry showed that the glass transition temperature (T(g)) for (poly-L-lactide)-PLLA, poly(L,D-lactide)-PDLLA, poly(lactide-co-glycolide)-PLGA and poly(lactide-co-caprolactone)-PLCL was 63 °C, 53 °C, 49 °C and 15 °C, respectively. Tensile stress-strain tests indicated that PLLA, PLGA, and PDLLA had a glassy behaviour when tested below T(g). The obtained Young modulus were 1477 MPa, 1330 MPa, 1306 MPa, and 9.55 MPa for PLLA, PLGA, PDLLA, and PLCL, respectively. Atomic force microscopy results confirmed that SbpA recrystallized on every polymer substrate exhibiting the native S-layer P4 lattice (a = b = 13 nm, γ = 90°). However, the polymer substrate influenced the domain size of the S-protein crystal, with the smallest size for PLLA (0.011 µm(2)), followed by PDLLA (0.034 µm(2)), and PLGA (0.039 µm(2)), and the largest size for PLCL (0.09 µm(2)). quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated that the adsorbed protein mass per unit area (~1800 ng cm(-2)) was independent of the mechanical, thermal, and crystalline properties of the polymer support. The slowest protein adsorption rate was observed for amorphous PLCL (the polymer with the weakest mechanical properties and lowest T(g)). QCM-D also monitored protein self-assembly in solution and confirmed that S-layer formation takes place in three main steps: adsorption, self-assembly, and crystal reorganization. Finally, this work shows that biodegradable polylactide derivatives films are a suitable support to form robust biomimetic S-protein layers.

Influence of surface chemistry and protein concentration on the adsorption rate and S-layer crystal formation.

Bacterial crystalline surface layers (S-layers) are the outermost envelope of prokaryotic organisms representing the simplest biological membranes developed during evolution. In this context, the bacterial protein SbpA has already shown its intrinsic ability to reassemble on different substrates forming protein crystals of square lattice symmetry. In this work, we present the interaction between the bacterial protein SbpA and five self-assembled monolayers carrying methyl (CH(3)), hydroxyl (OH), carboxylic acid (COOH) and mannose (C(6)H(12)O(6)) as functional groups. Protein adsorption and S-layer formation have been characterized by atomic force microscopy (AFM) while protein adsorption kinetics, mass uptake and the protein layer viscoelastic properties were investigated with quartz crystal microbalance with dissipation monitoring (QCM-D). The results indicate that the protein adsorption rate and crystalline domain area depend on surface chemistry and protein concentration. Furthermore, electrostatic interactions tune different protein rate adsorption and S-layer recrystallization pathways. Electrostatic interactions induce faster adsorption rate than hydrophobic or hydrophilic interactions. Finally, the shear modulus and the viscosity of the recrystallized S-layer on CH(3)C(6)S, CH(3)C(11)S and COOHC(11)S substrates were calculated from QCM-D measurements. Protein-protein interactions seem to play a main role in the mechanical stability of the formed protein (crystal) bilayer.

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, gamma = 90 degrees ), 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 microm(2) for APTS and 0.05 microm(2) for OTS are formed, while for silicon dioxide the protein domains have a typical size of about 32 microm(2). 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.