title: High-affinity tags fused to s-layer proteins probed by atomic force microscopy.
abstract: Two-dimensional, crystalline bacterial cell surface layers, termed S-layers, are one of the most commonly observed cell surface structures of prokaryotic organisms. In the present study, genetically modified S-layer protein SbpA of Bacillus sphaericus CCM 2177 carrying the short affinity peptide Strep-tag I or Strep-tag II at the C terminus was used to generate a 2D crystalline monomolecular protein lattice on a silicon surface. Because of the genetic modification, the 2D crystals were addressable via Strep-tag through streptavidin molecules. Atomic force microscopy (AFM) was used to investigate the topography of the single-molecules array and the functionality of the fused Strep-tags. In high-resolution imaging under near-physiological conditions, structural details such as protein alignment and spacing were resolved. By applying molecular recognition force microscopy, the Strep-tag moieties were proven to be fully functional and accessible. For this purpose, streptavidin molecules were tethered to AFM tips via approximately 8-nm-long flexible polyethylene glycol (PEG) linkers. These functionalized tips showed specific interactions with 2D protein crystals containing either the Strep-tag I or Strep-tag II, with similar energetic and kinetic behavior in both cases.
authors.affiliation: Institute of Biophysics, Johannes Kepler University of Linz, Linz, Austria.
authors.forename: Jilin
authors.initials: J
authors.lastname: Tang
authors.forename: Andreas
authors.initials: A
authors.lastname: Ebner
authors.forename: Nicola
authors.initials: N
authors.lastname: Ilk
authors.forename: Helga
authors.initials: H
authors.lastname: Lichtblau
authors.forename: Carina
authors.initials: C
authors.lastname: Huber
authors.forename: Rong
authors.initials: R
authors.lastname: Zhu
authors.forename: Dietmar
authors.initials: D
authors.lastname: Pum
authors.forename: Micheal
authors.initials: M
authors.lastname: Leitner
authors.forename: Vassili
authors.initials: V
authors.lastname: Pastushenko
authors.forename: Hermann J
authors.initials: HJ
authors.lastname: Gruber
authors.forename: Uwe B
authors.initials: UB
authors.lastname: Sleytr
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
mesh_terms: D003460:Crystallization; D008562:Membrane Glycoproteins / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D012825:Silicon / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D013499:Surface Properties
chemical_list: D008562:Membrane Glycoproteins; C055764:S-layer proteins; D011092:Polyethylene Glycols; D019809:Streptavidin; D012825:Silicon