Journal of Biological Chemistry
Volume 286, Issue 31, 5 August 2011, Pages 27416-27424
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Protein Structure and Folding
Single-molecule Force Spectroscopy Reveals the Individual Mechanical Unfolding Pathways of a Surface Layer Protein*

https://doi.org/10.1074/jbc.M111.251322Get rights and content
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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 microscopy-based 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.

Cell Surface Protein
Ligand Binding Protein
Protein Domains
Protein Stability
Protein Structure
Protein Unfolding
Single-molecule Force Spectroscopy
Surface Layer Protein

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*

This work was supported by the Air Force projects FA9550-09-1-0342, and FA9550-10-1-0223, by the Austrian Science Fund Projects P19047-B12 and P21954-B20 (to C. S.), and by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation, and Technology (BMVIT), the Styrian Business Promotion Agency, the Standortagentur Tirol, and Technology Agency of the City of Vienna through the COMET Funding Program managed by the Austrian Research Promotion Agency FFG.

1

Holds a DOC-fFORTE fellowship of the Austrian Academy of Sciences.