High-affinity tags fused to s-layer proteins probed by atomic force microscopy.
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.
Tang
Jilin
J
Institute of Biophysics, Johannes Kepler University of Linz, Linz, Austria.
Ebner
Andreas
A
Ilk
Nicola
N
Lichtblau
Helga
H
Huber
Carina
C
Zhu
Rong
R
Pum
Dietmar
D
Leitner
Micheal
M
Pastushenko
Vassili
V
Gruber
Hermann J
HJ
Sleytr
Uwe B
UB
Hinterdorfer
Peter
P
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
D008562:Membrane Glycoproteins; C055764:S-layer proteins; D011092:Polyethylene Glycols; D019809:Streptavidin; D012825:Silicon