Single-molecule Force Spectroscopy Reveals the Individual Mechanical Unfolding Pathways of a Surface Layer Protein

Abstract

Surface layers (S-layers) represent an almost universal feature of archaeal cell envelopes and are probably the most abundant bacterial cell proteins. S-layers are monomolecular crystalline structures of single protein or glycoprotein monomers that completely cover the cell surface during all stages of the cell growth cycle, thereby performing their intrinsic function under a constant intra-and intermolecular mechanical stress. In Gram-positive bacteria, the individual S-layer proteins are anchored by a specific binding mechanism to polysaccharides (secondary cell wall polymers) that are linked to the underlying peptidoglycan layer. In this work, atomic force microscopybased single-molecule force spectroscopy and a polyprotein approach are used to study the individual mechanical unfolding pathways of an S-layer protein. We uncover complex unfolding pathways involving the consecutive unfolding of structural intermediates, where a mechanical stability of 87 pN is revealed. Different initial extensibilities allow the hypothesis that S-layer proteins adapt highly stable, mechanically resilient conformations that are not extensible under the presence of a pulling force. Interestingly, a change of the unfolding pathway is observed when individual S-layer proteins interact with secondary cell wall polymers, which is a direct signature of a conformational change induced by the ligand. Moreover, the mechanical stability increases up to 110 pN. This work demonstrates that single-molecule force spectroscopy offers a powerful tool to detect subtle changes in the structure of an individual protein upon binding of a ligand and constitutes the first conformational study of surface layer proteins at the single-molecule level.

S-layers: principles and applications

“…The crystal structure of rSbsC 31–844 (P2 1 space group symmetry) revealed a novel fold, consisting of six separate domains, which are connected by short flexible linkers. Furthermore, SCWP binding induced considerable stabilization of the N‐terminal domain (Pavkov et al ., ) what was later on accordingly confirmed for SbsB by AFM‐based single‐molecule spectroscopy (Horejs et al ., ). To complete the structure of the full‐length protein, additional soluble constructs containing the crucial domains for self‐assembly were cloned, expressed, and purified (Dordic et al ., ).…”

Agglutinating Secretory IgA Preserves Intestinal Epithelial Cell Integrity during Apical Infection by Shigella flexneri

“…The experimental setting was designed to study the effect of M90T-triggered damage after overnight infection, well beyond the time course usually accessible in in vivo models (13,22). Bacteria were initially found to infect a limited number of polarized cells, leading to preferential targeting of the tight junction's seal; such a feature has been described for S. flexneri serotype 2 and also for other pathogens, such as enterohemorrhagic Escherichia coli and Salmonella strains (8,9,39), yet at very high MOI that do not reflect the low doses sufficient to infect the human gut. Over time, the progressive overwhelming proliferation of S. flexneri induced irreversible damage to the cell architecture, as reflected by the complete depolymerization of actin fibers.…”

Atomic force microscopy: A nanoscopic view of microbial cell surfaces

“…In SMFS the molecule of interest is tethered to the AFM tip via distensible crosslinkers and the complimentary pair to the substrate (Francius et al, 2009). On retraction of the tip from the surface, the forces involved in the unbinding can be monitored in real time (Horejs et al, 2011). The force curve recorded in SMFS often shows a nonlinear elongation force, reflecting stretching of flexible molecules on the sample (and on the tip), until the adhesion 'pull-off' force is observed.…”