From Native to Non‐Native Two‐Dimensional Protein Lattices through Underlying Hydrophilic/Hydrophobic Nanoprotrusions

Abstract

Protein crystallization is a crucial step in the study of protein structure and function, [1] as well as in biosensing. [2][3][4] In many cases, it proceeds under ill-controlled conditions, which make it difficult to predict the outcome or feasibility. On the other hand, controlled two-dimensional (2D) protein recrystallization has been mainly accomplished through metal-ion coordination to accessible histidine residues [5] or specific interactions with high-affinity ligands.[6] Both approaches, however, involve knowledge of the protein structure and, in some cases, protein engineering. In this work, we show that control of the morphology of a 2D wild-type protein crystal is possible by using a chemical nanotuner as a substrate. In this way, nonspecific substrate-protein interactions can be finely modulated to select the self-assembly pathway to the corresponding protein crystal. Our model system consists of a bacterial S-layer, a two-dimensional (glyco)protein crystal located in the cell wall of many prokaryotic organisms.[7] The S-layers act as selective, protective barriers that mediate cell development.[8] The chemical nanotuner is a set of selfassembled monolayers (SAMs) of dialkyldisulfide derivatives with the formula CH 3 (CH 2 ) 11+m SS(CH 2 ) 11 OH, in which m is the chain-length difference in methylene units between the methyl-and the hydroxy-terminated branches.[9] The resulting surface is composed of OH-and CH 3 -terminated thiolates in a stand-up conformation with a defined chemical functionality ratio of 50 %. These SAMs contain submolecular protrusions whose nature is controlled by the m value: m < 0 indicates that the protruding chains are OH terminated, whereas m > 0 denotes that the surface protrusions are methyl functionalized (Figure 1). The variation in the m value determines the protein-recrystallization route.

In Vitro Characterization of the Two-Stage Non-Classical Reassembly Pathway of S-Layers

“…A major breakthrough in the characterization of S-layer lattice formation was facilitated by the introduction of atomic force microscopy (AFM; also often referred to as scanning force microscopy (SFM)) since then it was not only possible to image S-layer lattices on technologically relevant substrates such as silicon wafers or glass but—in particular—to investigate the interactions and assembly dynamics in real time [17,18,19,20,21,22,23]. In a seminal work, De Yoreo and co-workers were able to elucidate the non-classical pathway of crystal growth of the S-layer protein SbpA from Lysinibacillus sphaericus ATCC 4525 (ATCC; American type Culture Collection) on supported lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphocholine (POPC) deposited on mica substrates [21].…”

Fluorescence energy transfer in the bi-fluorescent S-layer tandem fusion protein ECFP–SgsE–YFP

“…Molecular dynamics simulations have been used to elucidate the structure of the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 (Horejs et al, 2008), whilst crystallisation studies have helped to gain insight into different parts of the S-layer protein SbsC from G. stearothermophilus ATCC 12980 (Pavkov et al, 2008). Self-assembly studies on solid supports have revealed the importance of the surface chemistry for the protein layer structure of SbpA from Lysinibacillus sphaericus CCM 2177 as well as for the formation kinetics (Moreno-Flores et al, 2008;Eleta Lopez et al, 2010).…”

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

“…They are fairly similar to the type of polymer substrate and agree to the ones observed on native S-layers, 15 and for many different kinds of substrates. [20][21][22][23][24]32 The AFM images ( Figure 5) show that these layers are characterized by protein crystal domains which do not differ in lattice parameters but in the lattice orientation. As can be observed, larger protein domains appear on amorphous poly- mers (PDLLA, PLGA, and PLCL) than on semicrystalline PLLA.…”

“…In previous works, the influence of the hydrophobicity on the recrystallization process of SbpA has been reported. 21,23 On hydrophilic silicon surfaces, large crystal domains are formed, while on hydrophobic silanecoated substrates, the domain sizes are 100 times smaller. 23 The biodegradable polymers presented in this work have contact angles within a small range of 80 • -83 • (Table V).…”

“…[15][16][17][18] These biosupramolecular structures have already been studied in a wide range of environments and solid suports. [19][20][21][22][23][24] Moreover, S-layer technology has moved on in the last decade in the synthesis and development of S-layer fusion proteins, which represents a big step in nanobiotechnology 25,26 and constitutes a promising field in surface 2D nanofunctionalization. [27][28][29][30][31] The aim of our work was to investigate the interaction between some of the varieties of lactic acid based polymers of different physicochemical properties (e.g., crystallinity, mechanical properties, etc.)…”