Electrochemical-QCMD Control over S-Layer (SbpA) Recrystallization with Fe2+ as Specific Ion for Self-Assembly Induction

Abstract

The critical role of divalent ions (M²+) in the self-assembly of SbpA S-layer proteins (from Lysinibacillus sphaericus CCM 2177) into crystalline structures has been reported in several studies. Hence, ions such as magnesium, barium, nickel and, most commonly, calcium (Ca²+) have proven to trigger both protein-protein and protein-substrate interactions involved in the two-stage non-classical pathway recrystallization followed by SbpA units. As a result, two dimensional, crystalline nanometric sheets in a highly ordered tetrameric state (p4) can be formed on top of different surfaces. The use of iron in its ferrous state (Fe2+) as self-assembly inducing candidate has been omitted so far due to its instability under aerobic conditions, tending to natural oxidation to the ferric (Fe3+) state. In this work, the potentiality of assembling fully functional S-layers from iron (II) salts (FeCl2 and FeSO4) is described for the first time. A combination of chemical (oxidation retardants) and electrical (−1 V potential) factors has been applied to effectively act against such an oxidizing trend. Formation of the respective crystalline films has been followed by means of Electrochemical Quartz Crystal Microbalance with Dissipation (EQCM-D) measurements and complementary Atomic Force Microscopy (AFM) topography studies, which prove the presence of squared lattice symmetry at the end of the recrystallization process. Both techniques, together with additional electrochemical tests performed over the ion permeability of both types of S-layer coatings formed, show the influence of the counterion chosen (chloride vs. sulphate) in the final packing and performance of the S-layer. The presence of an underlying Secondary Cell Wall Polymer (SCWP) as in the natural case contributes to pair both systems, due to the high lateral motility freedom provided by this biopolymer to SbpA units in comparison to uncoated substrates.

Life under Continuous Streaming: Recrystallization of Low Concentrations of Bacterial SbpA in Dynamic Flow Conditions

“…Overall, the role of the experimental conditions applied has been again shown to induce a relevant impact on the assembly and recrystallization of SbpA bacterial protein S-layers. This was already described for factors such as the substrate wettability, substrate active volume, or even other types of divalent cations [19,21,26]. In this case, constant protein feeding allows lowering the protein bulk concentration required to form a homogeneous and well-packed S-layer where the featuring crystallinity can still be present, especially if compared to experiments performed in stopped-flow conditions.…”

“…The nanostructure of the protein layer is responsible for some of the practical properties of these biomimetic films, like its described non-fouling activity [16] of high technological usefulness [17,18]. In recent years, several studies have addressed some general questions from the assembly and recrystallization of SbpA; namely, the influence of the substrate wettability [19] or the surface chemistry [20], and the use of alternative divalent cations [21]. Recent studies have provided a more dynamic insight on the SbpA S-layer formation process in order to develop optimal crystallization models.…”