crystals Article Crystalline S-Layer Protein Monolayers Induce Water Turbulences on the Nanometer Scale Rupert Tscheliessnig 1, *, Andreas Breitwieser 2 , Uwe B. Sleytr 3 and Dietmar Pum 2   Citation: Tscheliessnig, R.; Breitwieser, A.; Sleytr, U.B.; Pum, D. Crystalline S-Layer Protein Monolayers Induce Water Turbulences on the Nanometer Scale. Crystals 2021, 11, 1147. https://doi.org/10.3390/ cryst11091147 Academic Editors: Ryan Taoran Wang, Yi Feng, Ya-Dong Yu, Eliana B. Souto and Yan V. Zubavichus Received: 9 August 2021 Accepted: 14 September 2021 Published: 20 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department for Biotechnology, Institute of Bioprocess Science and Engineering, University of Natural Resources and Life Sciences, 1190 Vienna, Austria 2 Department for Nanobiotechnology, Institute for Biophysics, University of Natural Resources and Life Sciences, 1190 Vienna, Austria; andreas.breitwieser@boku.ac.at (A.B.); dietmar.pum@boku.ac.at (D.P.) 3 Department of Nanobiotechnology, Institute of Synthetic Bioarchitectures, University of Natural Resources and Life Sciences, 1190 Vienna, Austria; uwe.sleytr@boku.ac.at * Correspondence: rupert.tscheliessnig@boku.ac.at Abstract: Bacterial surface layers (S-layers) have been observed as the outermost cell envelope component in a wide range of bacteria and most archaea. They are one of the most common prokaryotic cell surface structures and cover the cells completely. It is assumed that S-layers provide selection advantages to prokaryotic cells in their natural habitats since they act as protective envelopes, as structures involved in cell adhesion and surface recognition, as molecular or ion traps, and as molecular sieves in the ultrafiltration range. In order to contribute to the question of the function of S-layers for the cell, we merged high-resolution cryo-EM and small-angle X-ray scattering datasets to build a coarse-grained functional model of the S-layer protein SbpA from Lysinibacillus sphaericus ATCC 4525. We applied the Navier–Stokes and the Poisson equations for a 2D section through the pore region in the self-assembled SbpA lattice. We calculated the flow field of water, the vorticity, the electrostatic potential, and the electric field of the coarse-grained model. From calculated local changes in the flow profile, evidence is provided that both the characteristic rigidity of the S-layer and the charge distribution determine its rheological properties. The strength of turbulence and pressure near the S-layer surface in the range of 10 to 50 nm thus support our hypothesis that the S-layer, due to its highly ordered repetitive crystalline structure, not only increases the exchange rate of metabolites but is also responsible for the remarkable antifouling properties of the cell surface. In this context, studies on the structure, assembly and function of S-layer proteins are promising for various applications in nanobiotechnology, biomimetics, biomedicine, and molecular nanotechnology. Keywords: S-layers; small angle X-ray scattering; cryo-EM; Navier–Stokes equation; Poisson equation; anti-fouling 1. Introduction Crystalline bacterial surface layers (called S-layers) are known to be one of the most common cell surface structures in archaea and bacteria [1–5]. S-layers are monomolecular arrays of a single protein or glycoprotein species (M w 40 to 200 kDa) and completely cover the archaeal or bacterial cell (Figure 1). Furthermore, S-layer proteins can be considered one of the most abundant biopolymers on earth since the biomass of prokaryotic organisms exceeds that of eukaryotic organisms [6]. S-layers exhibit either oblique (p1, p2), square (p4) or hexagonal (p3, p6) lattice symmetry. Accordingly, a unit cell (morphological unit) consists of one, two, four, three, or six identical monomers. Figure 1 shows a transmission electron microscopy (TEM) image of a bacterial cell with an S-layer with square lattice symmetry. The unit cell dimensions of S-layers range from 3 to 30 nm, while the thickness is between 5 and 10 nm (up to 70 nm in archaea). Due to their crystalline nature, S-layers are porous protein networks (30–70% porosity) with pores of uniform size (2–8 nm) and morphology [7,8]. Crystals 2021, 11, 1147. https://doi.org/10.3390/cryst11091147 https://www.mdpi.com/journal/crystals