Cysteine engineering of polyproteins for single-molecule force spectroscopy

Hendrik Dietz, Morten Bertz, Michael Schlierf, Felix Berkemeier, Thomas Bornschlögl, Jan Philipp Junker, Matthias Rief

Research output: Contribution to journalArticle

52 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)80-84
Number of pages5
JournalNature Protocols
Volume1
Issue number1
DOIs
Publication statusPublished - 2006 Jun
Externally publishedYes

Fingerprint

Polyproteins
Cysteine
Spectroscopy
Molecules
Proteins
Single Molecule Imaging
Mechanics
Polymerization
Stretching
Spectrum Analysis
Microscopes
Topology
Amino Acids
Mechanical properties

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Dietz, H., Bertz, M., Schlierf, M., Berkemeier, F., Bornschlögl, T., Junker, J. P., & Rief, M. (2006). Cysteine engineering of polyproteins for single-molecule force spectroscopy. Nature Protocols, 1(1), 80-84. https://doi.org/10.1038/nprot.2006.12

Cysteine engineering of polyproteins for single-molecule force spectroscopy. / Dietz, Hendrik; Bertz, Morten; Schlierf, Michael; Berkemeier, Felix; Bornschlögl, Thomas; Junker, Jan Philipp; Rief, Matthias.

In: Nature Protocols, Vol. 1, No. 1, 06.2006, p. 80-84.

Research output: Contribution to journalArticle

Dietz, H, Bertz, M, Schlierf, M, Berkemeier, F, Bornschlögl, T, Junker, JP & Rief, M 2006, 'Cysteine engineering of polyproteins for single-molecule force spectroscopy', Nature Protocols, vol. 1, no. 1, pp. 80-84. https://doi.org/10.1038/nprot.2006.12
Dietz H, Bertz M, Schlierf M, Berkemeier F, Bornschlögl T, Junker JP et al. Cysteine engineering of polyproteins for single-molecule force spectroscopy. Nature Protocols. 2006 Jun;1(1):80-84. https://doi.org/10.1038/nprot.2006.12
Dietz, Hendrik ; Bertz, Morten ; Schlierf, Michael ; Berkemeier, Felix ; Bornschlögl, Thomas ; Junker, Jan Philipp ; Rief, Matthias. / Cysteine engineering of polyproteins for single-molecule force spectroscopy. In: Nature Protocols. 2006 ; Vol. 1, No. 1. pp. 80-84.
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