Detection of metal binding sites on functional S-layer nanoarrays using single molecule force spectroscopy.
Crystalline bacterial cell surface layers (S-layers) show the ability to recrystallize into highly regular pattern on solid supports. In this study, the genetically modified S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177, carrying a hexa-histidine tag (His(6)-tag) at the C-terminus, was used to generate functionalized two-dimensional nanoarrays on a silicon surface. Atomic force microscopy (AFM) was applied to explore the topography and the functionality of the fused His(6)-tags. The accessibility of the His(6)-tags was demonstrated by in-situ anti-His-tag antibody binding to the functional S-layer array. The metal binding properties of the His(6)-tag was investigated by single molecule force microscopy. For this purpose, newly developed tris-NTA was tethered to the AFM tips via a flexible polyethylene glycol (PEG) linker. The functionalized tips showed specific interactions with S-layer containing His(6)-tags in the presence of nickel ions. Thus the His(6)-tag is located at the outer surface of the S-layer and can be used for stable but reversible attachment of functional tris-NTA derivatives.
Tang
Jilin
J
Institute of Biophysics, Johannes Kepler University of Linz, Austria.
Ebner
Andreas
A
Kraxberger
Bernhard
B
Leitner
Michael
M
Hykollari
Alba
A
Kepplinger
Christian
C
Grunwald
Christian
C
Gruber
Hermann J
HJ
Tampé
Robert
R
Sleytr
Uwe B
UB
Ilk
Nicola
N
Hinterdorfer
Peter
P
D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D008562:Membrane Glycoproteins / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D036103:Nanotechnology / Q000379:methods*
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; C055764:S-layer proteins