Absorption, steady-state fluorescence, fluorescence lifetime, and 2D self-assembly properties of engineered fluorescent S-layer fusion proteins of Geobacillus stearothermophilus NRS 2004/3a.
S-layer fusion protein technology was used to design four different fluorescent fusion proteins with three different GFP mutants and the red fluorescent protein mRFP1. Their absorption spectra, steady-state fluorescence, and fluorescence lifetime were investigated as a function of pH. It was found that fluorescence intensities and lifetime of the GFP mutant S-layer fusion proteins decreased about 50% between pH 6 and pH 5. The spectral properties of the red S-layer fusion protein were minimally affected by pH variations. These results were compared with His-tagged reference fluorescent proteins, demonstrating that the S-layer protein did not change the general spectral properties of the whole fusion protein. In addition, the pK(a) values of the fluorescent S-layer fusion proteins were calculated. Finally, it was shown that the S-layer fusion proteins were able to self-assemble forming 2D nanostructures of oblique p2 symmetry with lattice parameters of about a = 11 nm, b = 14 nm, and gamma = 80 degrees . The fluorescence tag did not hinder the natural self-assembly process of the S-layer protein. The combination of the fluorescence properties and the self-assembly ability of the engineered fusion proteins make them a promising tool to generate biomimetic surfaces for future applications in nanobiotechnology at a wide range of pH.
Kainz
Birgit
B
Department for NanoBiotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 11, A-1190 Vienna, Austria.
Steiner
Kerstin
K
Möller
Marco
M
Pum
Dietmar
D
Schäffer
Christina
C
Sleytr
Uwe B
UB
Toca-Herrera
José L
JL
D000042:Absorption; D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure; D005453:Fluorescence; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000648:ultrastructure; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000648:ultrastructure
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins