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Cysteine engineering of polyproteins for single-molecule force spectroscopy

Abstract

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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Figure 1
Figure 2: Dispersion of polymeric lengths in an 0.2-mM GFP(3, 212) polyprotein solution after 80 h of incubation at 37 °C.

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Acknowledgements

This work has been supported by an SFB413 grant of the Deutsche Forschungsgemeinschaft.

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H.D. developed the protocol, performed and designed research, analyzed the data and wrote the manuscript. M.B. performed size-exclusion chromatography and gel electrophoresis experiments and analyzed the data from these experiments. F.B. performed supporting experiments on GFP polyproteins. M.S., T.B., J.P.J. helped to refine the protocol. M.R. designed research and wrote the manuscript. All authors commented on the manuscript.

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Correspondence to Hendrik Dietz.

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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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