The Flavoprotein Dodecin as a Redox Probe for Electron Transfer through DNA

Abstract

Flavin away: Dodecin binds oxidized flavins, whereas reduction of the bound flavin induces dissociation of the holoprotein into apododecin and free flavins. The stepwise reconstitution of dodecin on flavin‐terminated ds‐DNA monolayers showed that although electrochemical flavin reduction (i.e. electron transfer through DNA) was not possible, apododecin (gray circles) could be released by chemical reduction (see scheme).

Nanomechanical properties of protein–DNA layers with different oligonucleotide tethers

“…by π-stacking interaction between the two isoalloxazine moieties of the flavins and the two tryptophans W36 belonging to the apoprotein. 2,5 This results in a relatively low value of k off = 1.9 × 10 –4 s –1 . Since for the second binding event not only k on but also k off is smaller than for the first, the value of K d for the second binding/unbinding event ( K d = 2.7 × 10 –7 M) is similar to that of the first ( K d = 1.4 × 10 –7 M).…”

“…This assumption is made based on crystallographic data from apododecin (with empty pockets) and dodecin complexes with different flavin ligands, which show little to no difference between Cα-atom positions. 2–5 Thus the (apo)dodecin binding pockets comprise a fixed steric configuration and there is nearly no structural rearrangement of the complex upon ligand binding. Ligands can enter only if they approach the binding pocket in a precise orientation.…”

“…For QCM-D and SPFS measurements ssDNA comprising 20 bases (5′-AAC-TAC-TGG-GCC-ATC-GTG-AC-3′) modified at the 5′-end with three dithiane groups introduced by the use of dithiol-phosphoramidite (6750.6 g mol –1 ), as well as complementary ssDNA modified with a flavin (CofC4) at the 5′-end (6472.4 g mol –1 ) were used. 1,5 Moreover, ssDNA (5′-GTC-ACG-ATG-GCC-CAG-TAG-TT-3′) modified with a flavin (CofC4) at the 5′ end and one dithiane ring at the 3′ end was purchased (6672.4 g mol –1 ). For SPR, SPFS, and QCM-D measurements ssDNA (5′-AAC-TAC-TGG-GCC-ATC-GTG-AC-3′) labeled with the fluorescent dye Cy5® at the 5′ end was hybridized with complementary flavin-modified ssDNA mentioned above to form a monodentate Cy5® labeled flavin-dsDNA ligand.…”

“…These protein layers were further modified by adsorption of bidentate flavin ligands resulting in stable sandwich-type DNA–protein–DNA nanostructures. Since dodecin binds flavin ligands with high affinity when they are oxidized, whereas flavin reduction induces the dissociation of the holoprotein in apododecin and free flavin ligands, 2,5,7 the generated nanostructures could be disassembled by chemical reduction using a buffered solution of sodium dithionite and reprogrammed by adsorption of a different type of bidentate flavin anchored ligand after reassembly of the dodecin layer. 1 Based on this previous study apododecin–flavin could evolve into an interesting alternative to streptavidin–biotin, which is probably the most widely applied multi-ligand binding system for surface modification.…”

“…While in previous studies regarding the reconstitution of dodecin on surfaces always dsDNA tethers comprising 20 base pairs (bp) were used, 1,2,5 here a detailed QCM-D study is presented showing how the length and flexibility of the surface grafted and flavin-terminated oligonucleotide tethers influence the stability and the viscoelastic properties of the resulting DNA–protein layers, which may then be used to bind additional bi- or multidentate ligands. In addition differences in the binding and unbinding behavior of flavin-dsDNA and flavin-ssDNA ligands are measured by QCM-D and surface plasmon fluorescence spectroscopy (SPFS).…”