De novo design, synthesis, and characterization of quinoproteins |
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Authors: | Li Wen-Wu Hellwig Petra Ritter Michaela Haehnel Wolfgang |
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Affiliation: | Institut für Biologie II/Biochemie, Albert-Ludwigs-Universit?t Freiburg, Sch?nzlestrasse 1, 79104 Freiburg, Germany. wenwu.li@biologie.uni-freiburg.de |
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Abstract: | Quinones and quinoproteins are essential redox components and enzymes in biological systems. Here, we report the de novo design, synthesis, and properties of model four-alpha-helix bundle quinoproteins. The proteins were designed and constructed from three different helices with 21 or 22 amino acid residues by chemoselective ligation to a cyclic decapeptide template. A free cysteine unit is placed at the hydrophobic core of the protein for binding of ubiquinone-0 and menaquinone-0 through a thioether bond. The quinoproteins with molecular weights of 11-12 kDa were characterized by electrospray ionization mass spectrometry, UV/Vis spectroscopy, size-exclusion chromatography, circular dichroism measurements, (1)H NMR spectroscopy, cyclic voltammetry, and redox-induced FTIR difference spectroscopy. The midpoint redox potentials at pH 8 in aqueous solution E(m,8) of thioether conjugates with N-acetyl cysteine methyl ester were 89 mV and -63 mV and with a synthetic protein 229 mV and 249 mV versus standard hydrogen electrode (SHE) for ubiquinone-0 and menaquinone-0, respectively. Detailed redox-induced FTIR difference spectroscopic studies of the model compounds and quinoproteins show the special resonance features for C=O bands at 1656-1660 and 1655-1665 cm(-1) due to the sulfur substitution to ubiquinone-0 and menaquinone-0, respectively. The construction of model quinoproteins represents a significant step toward more complex artificial redox systems. |
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Keywords: | cyclic voltammetry IR spectroscopy protein design protein structures quinones |
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