title: 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.
authors.affiliation: Physik Department, Technische Universität München, James-Franck-Strasse, D-85748 Garching bei München, Germany. dietz@ph.tum.de
authors.forename: Hendrik
authors.initials: H
authors.lastname: Dietz
authors.forename: Morten
authors.initials: M
authors.lastname: Bertz
authors.forename: Michael
authors.initials: M
authors.lastname: Schlierf
authors.forename: Felix
authors.initials: F
authors.lastname: Berkemeier
authors.forename: Thomas
authors.initials: T
authors.lastname: Bornschlögl
authors.forename: Jan Philipp
authors.initials: JP
authors.lastname: Junker
authors.forename: Matthias
authors.initials: M
authors.lastname: Rief
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D020815:Polyproteins / Q000737:chemistry / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D013057:Spectrum Analysis
chemical_list: D020815:Polyproteins; D011994:Recombinant Proteins; D003545:Cysteine