Electrochemical switching of the flavoprotein dodecin at gold surfaces modified by flavin-DNA hybrid linkers

Abstract

Dodecin from Halobacterium salinarum is a dodecameric, hollow-spherical protein, which unspecifically adopts flavin molecules. Reduction of flavin dodecin holocomplexes induces dissociation into apododecin and free flavin. Unspecific binding and dissociation upon reduction were used as key properties to construct an electrochemically switchable surface, which was able to bind and release dodecin apoprotein depending on the applied potential. A flavin modified electrode surface ͑electrode-DNA-flavin͒ was generated by direct adsorption of double stranded DNA ͑ds-DNA͒ equipped with flavin and disulfide modifications at opposite ends. While the disulfide functionality enabled anchoring the ds-DNA at the gold surface, the flavin exposed at the surface served as the redox-active dodecin docking site. The structures of protein and flavin-DNA hybrid ligands were optimized and characterized by x-ray structural analysis of the holocomplexes. By surface plasmon resonance ͑SPR͒ spectroscopy, the adsorption of flavin modified DNA as well as the binding and the electrochemically induced release of dodecin apoprotein could be shown. When the surface immobilization protocol was changed from direct immobilization of the modified ds-DNA to a protocol, which included the hybridization of flavin and thiol modified DNA at the surface, the resulting monolayer was electrochemically inactive. A possible explanation for the strong influence of the surface immobilization protocol on addressing dodecin by the applied potential is that electron transfer is rather mediated by defects in the monolayer than modified ds-DNA.

Blue‐Light‐Triggered Photorelease of Active Chemicals Captured by the Flavoprotein Dodecin

“…The increased holocomplex stabilities when lumichrome is added as a second ligand come from the preference of dodecin for binding flavin/ lumichrome heterodimers. [14] Besides the intrinsic higher stability of the resulting mixed aromatic tetrad, saturation of the binding positions adjacent to bulky ligands with the small lumichrome might reduce steric restraints between the ligand and the dodecin apoprotein and additionally promote high-affinity binding. In Figure 2, X-ray structures of the tE-mutated [14] apododecin, tE dodecin with bound ligand 1, and E45A-mutated dodecin with bound ligand 2 are shown.…”

“…Based on the dodecin characteristic of only binding oxidized flavins with high affinity, reduction of the flavin can be used to trigger the dissociation of the holoprotein into A C H T U N G T R E N N U N G apoprotein and free ligands. [14] As flavoproteins can be reduced photochemically by irradiation with blue light in the presence of EDTA or other suitable electron donors, [15,16] we indented to use dodecin for establishing a blue-light-triggered photorelease system (Scheme 1). Any drug or active compound can be captured by dodecin after it has been linked to a flavin anchoring group.…”

“…Consequently, the loss of the H-bond interactions between the aliphatic ribityl chain and the protein envelope (E45 and V35) only marginally decreases ligand affinities. [14] In this approach, the artificial flavin CofC6, which has a -(CH 2 ) 6 -NH 2 group as an aliphatic moiety, was used as an anchor to lock active molecules to A C H T U N G T R E N N U N G dodecin. Based on the dodecin characteristic of only binding oxidized flavins with high affinity, reduction of the flavin can be used to trigger the dissociation of the holoprotein into A C H T U N G T R E N N U N G apoprotein and free ligands.…”

“…[22,23] ET from EDTA and tryptophan are competitive processes, but in contrast to ET from EDTA, which is followed by the release of CO 2 , [15,16] ET from tryptophan is reversible. Next, the photochemical reduction of dodecin saturated with an artificial flavin termed CofC6 (a flavin bearing a hexylamino substituent at the N(10) position of the isoalloxazine moiety) [14] and dodecin binding the flavin-DNA ligand 1 were compared (see Figure 3). The absorption bands of the oxidized flavins with maxima at about 360 and 440 nm decrease upon ongoing irradiation.…”

“…[14] Oligomeric DNA consisting of five bases and a blue BDP dye (purchased from Molecular Probes) were chosen as models representing different types of drugs, reagents, or active compounds.…”