Life under continuous streaming: Recrystallization of low concentrations of bacterial SbpA in dynamic flow conditions

Abstract

The well-known bacterial S-layer protein SbpA from Lysinibacillus sphaericus CCM2177 induces spontaneous crystal formation via cooperative self-assembly of the protein subunits into an ordered supramolecular structure. Recrystallization occurs in the presence of divalent cations (i.e., Ca2+) and finally leads to producing smooth 2-D crystalline coatings composed of squared (p4) lattice structures. Among the factors interfering in such a process, the rate of protein supply certainly plays an important role since a limited number of accessible proteins might turn detrimental for film completion. Studies so far have mostly focused on high SbpA concentrations provided under stopped-flow or dynamic-flow conditions, thus omitting the possibility of investigating intermediate states, in which dynamic flow is applied for more critical concentrations of SbpA (i.e., 25, 10, and 5 μg/mL). In this work, we have characterized both physico-chemical and topographical aspects of the assembly and recrystallization of SbpA protein in such low concentration conditions by means of in situ Quartz Crystal Microbalance with Dissipation (QCMD) and atomic force microscopy (AFM) measurements, respectively. On the basis of these experiments, we can confirm how the application of a dynamic flow influences the formation of a closed and crystalline protein film from low protein concentrations (i.e., 10 μg/mL), which otherwise would not be formed.