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. 2018 Jul 11:6:278.
doi: 10.3389/fchem.2018.00278. eCollection 2018.

Selective Targeting of Proteins by Hybrid Polyoxometalates: Interaction Between a Bis-Biotinylated Hybrid Conjugate and Avidin

Valeria A Zamolo  1 , Gloria Modugno  1 , Elisa Lubian  1 , Alessandro Cazzolaro  1 , Fabrizio Mancin  1 , Livia Giotta  2 , Disma Mastrogiacomo  2 , Ludovico Valli  2 , Alessandra Saccani  3 , Silke Krol  3   4 , Marcella Bonchio  1 , Mauro Carraro  1
Affiliations

Selective Targeting of Proteins by Hybrid Polyoxometalates: Interaction Between a Bis-Biotinylated Hybrid Conjugate and Avidin

Valeria A Zamolo et al. Front Chem. .

Abstract

The Keggin-type polyoxometalate [γ-SiW10O36]8- was covalently modified to obtain a bis-biotinylated conjugate able to bind avidin. Spectroscopic studies such as UV-vis, fluorimetry, circular dichroism, coupled to surface plasmon resonance technique were used to highlight the unique interplay of supramolecular interactions between the homotetrameric protein and the bis-functionalized polyanion. In particular, the dual recognition mechanism of the avidin encompasses (i) a complementary electrostatic association between the anionic surface of the polyoxotungstate and each positively charged avidin subunit and (ii) specific host-guest interactions between each biotinylated arm and a corresponding pocket on the tetramer subunits. The assembly exhibits peroxidase-like reactivity and it was used in aqueous solution for L-methionine methyl ester oxidation by H2O2. The recognition phenomenon was then exploited for the preparation of layer-by-layer films, whose structural evolution was monitored in situ by ATR-FTIR spectroscopy. Finally, cell tracking studies were performed by exploiting the specific interactions with a labeled streptavidin.

Keywords: avidin; bio-hybrids; biotin; oxidation catalysis; polyoxometalates; recognition; surface plasmon resonance.

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Figures

Figure 1
Figure 1
Structure of POM-biot2 [γ-SiW10O36{(C5H7N2OS)(CH2)4CONH(CH2)3Si}2O]4− showing the organic domain, including the biotin and an aminopropyl spacer, and the ball & stick model of the inorganic scaffold (blue balls = W atoms; red balls = oxygen atoms), and possible POM binding modes (bridging or tweezer) with the avidin homotetramer.
Figure 2
Figure 2
(Top) Fluorescence spectrum of avidin (1 × 10−6 M, in PBS 5% DMSO, 25°C) in the presence of increasing amount of TBA-POM-biot2 (0–6 μm) dissolved in PBS 5% DMSO. Excitation wavelength: 280 nm; 1 cm path length quartz cell. Inset: Stern-Volmer graphs, obtained for the fluorescence emission at 338 nm, in the presence of different POMs or Biot, in the range 0–2 μM. (Bottom) UV-vis spectra of tetrameric avidin-HABA (0.65 × 10−6 M, in HEPES buffer, 25°C) in the presence of increasing amount of biotin equivalents. Inset: % absorbance decrease observed at 500 nm in the presence of Biot (0–0.81 μM) or different POMs (0–0.41 μM, 1 biotin equiv. corresponding to 0.5 POM equivs.).
Figure 3
Figure 3
Sensorgram (red lines) obtained for TBA-POM-biot2 (Top) and of Na-POM-biot2 (Bottom) with their corresponding fittings obtained as the result of three concomitant events: a specific binding (gray track), a reversible interaction (purple curve) and a negligible bulk contribution arising from dilution artifacts (pink line). Conditions: flow (10 μl/min) of POM-biot2 solutions (5, 10, 20, 40 μM) in HBS-ES buffer (pH = 7), with 5% DMSO (only for TBA-POM-biot2) on an avid functionalized dextran-coated gold chip.
Figure 4
Figure 4
Layer by Layer deposition of alternating avidin/Na-POM-biot2 layers. The deposition model proposed (left) is in agreement with adsorption kinetics of both components (right). The letters A, B, C, D allow to relate the steady infrared signals with the molecular architecture of the films. Marker bands for avidin and POM are at 1,536 and 919 cm-1 respectively. Each kinetic curve was obtained in difference mode, thus following the deposition of the last fluxed component.
Figure 5
Figure 5
HeLa cells incubated at 37°C for 24 h in presence of 0.4 mg/mL of Na-POM-NH2 (Left) Na-POM-biot2 (Right), and stained with Atto 633-Streptavidin (red fluorescent signal). Nucleus stained by Hoechst 33342 (blue). Images acquired by a confocal light scanning microscope (CLSM). A similar signal was seen with a concentration of 0.2 mg/ml POM.
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