Single-molecule methods such as force spectroscopy give experimental access to the mechanical properties of protein molecules. So far, owing to the limitations of recombinant construction of polyproteins, experimental access has been limited to mostly the N-to-C terminal direction of force application. This protocol gives a fast and simple alternative to current recombinant strategies for preparing polyproteins. We describe in detail the method to construct polyproteins with precisely controlled linkage topologies, based on the pairwise introduction of cysteines into protein structure and subsequent polymerization in solution. Stretching such constructed polyproteins in an atomic force microscope allows mechanical force application to a single protein structure via two precisely controlled amino acid positions in the functional three-dimensional protein structure. The capability for site-directed force application can provide valuable information about both protein structure and directional protein mechanics. This protocol should be applicable to almost any protein that can be point mutated. Given correct setup of all necessary reagents, this protocol can be accomplished in fewer than 10 d.
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Bustamante, C., Chemla, Y.R., Forde, N.R. & Izhaky, D. Mechanical processes in biochemistry. Annu. Rev. Biochem. 73, 705–748 (2004).
Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M. & Gaub, H.E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997).
Li, H., Oberhauser, A.F., Fowler, S.B., Clarke, J. & Fernandez, J.M. Atomic force microscopy reveals the mechanical design of a modular protein. Proc. Natl. Acad. Sci. USA 97, 6527–6531 (2000).
Carrion-Vazquez, M. et al. Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering. Prog. Biophys. Mol. Biol. 74, 63–91 (2000).
Yang, G. et al. Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme. Proc. Natl. Acad. Sci. USA 97, 139–144 (2000).
Dietz, H. & Rief, M. Protein structure by mechanical triangulation. Proc. Natl. Acad. Sci. USA 103, 1244–1247 (2006).
Rounsevell, R.W., Steward, A. & Clarke, J. Biophysical investigations of engineered polyproteins: implications for force data. Biophys. J. 88, 2022–2029 (2005).
Li, L., Huang, H.H., Badilla, C.L. & Fernandez, J.M. Mechanical unfolding intermediates observed by single-molecule force spectroscopy in a fibronectin type III module. J. Mol. Biol. 345, 817–826 (2005).
Dietz, H. & Rief, M. Exploring the energy landscape of GFP by single-molecule mechanical experiments. Proc. Natl. Acad. Sci. USA 101, 16192–16197 (2004).
Fisher, T.E., Marszalek, P.E. & Fernandez, J.M. Stretching single molecules into novel conformations using the atomic force microscope. Nat. Struct. Biol. 7, 719–724 (2000).
Clausen-Schaumann, H., Seitz, M., Krautbauer, R. & Gaub, H.E. Force spectroscopy with single bio-molecules. Curr. Opin. Chem. Biol. 4, 524–530 (2000).
Zinober, R.C. et al. Mechanically unfolding proteins: the effect of unfolding history and the supramolecular scaffold. Protein Sci. 11, 2759–2765 (2002).
This work has been supported by an SFB413 grant of the Deutsche Forschungsgemeinschaft.
The authors declare no competing financial interests.
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Dietz, H., Bertz, M., Schlierf, M. et al. Cysteine engineering of polyproteins for single-molecule force spectroscopy. Nat Protoc 1, 80–84 (2006). https://doi.org/10.1038/nprot.2006.12
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