Reversible and Oriented Immobilization of Ferrocene-Modified Proteins

Abstract

Adopting supramolecular chemistry for immobilization of proteins is an attractive strategy that entails reversibility and responsiveness to stimuli. The reversible and oriented immobilization and micropatterning of ferrocene-tagged yellow fluorescent proteins (Fc-YFPs) onto β-cyclodextrin (βCD) molecular printboards was characterized using surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in combination with electrochemistry. The proteins were assembled on the surface through the specific supramolecular host-guest interaction between βCD and ferrocene. Application of a dynamic covalent disulfide lock between two YFP proteins resulted in a switch from monovalent to divalent ferrocene interactions with the βCD surface, yielding a more stable protein immobilization. The SPR titration data for the protein immobilization were fitted to a 1:1 Langmuir-type model, yielding K(LM) = 2.5 × 10(5) M(-1) and K(i,s) = 1.2 × 10(3) M(-1), which compares favorably to the intrinsic binding constant presented in the literature for the monovalent interaction of ferrocene with βCD self-assembled monolayers. In addition, the SPR binding experiments were qualitatively simulated, confirming the binding of Fc-YFP in both divalent and monovalent fashion to the βCD monolayers. The Fc-YFPs could be patterned on βCD surfaces in uniform monolayers, as revealed using fluorescence microscopy and atomic force microscopy measurements. Both fluorescence microscopy imaging and SPR measurements were carried out with the in situ capability to perform cyclic voltammetry and chronoamperometry. These studies emphasize the repetitive desorption and adsorption of the ferrocene-tagged proteins from the βCD surface upon electrochemical oxidation and reduction, respectively.

Redox-active host-guest supramolecular assemblies of peptides and proteins at surfaces

“…In this respect, we linked a yellow fluorescent protein (YFP) bearing a C-terminal thioester to a cysteine-functionalized ferrocene via expressed protein ligation (Fig. 5) [16,17]. We first exploited the interaction of these ferrocenylated YFPs with self-assembled monolayers (SAMs) of CB [7] (Fig.…”

“…A number of Fc-YFP constructs with different ferrocene valencies, (mono-and divalent) were generated by making use of the construct's intrinsic affinity for noncovalent homodimerization as well as cysteine residues for covalent homodimerization via the formation of disulfide bonds (Fig. 6) [17]. In short, one variant (Fc-dYFP) contained two point mutations that increased the affinity for noncovalent dimerization, while the so-called enhanced variant (Fc-eYFP) lacked these dimerization-promoting mutations [17].…”

“…6) [17]. In short, one variant (Fc-dYFP) contained two point mutations that increased the affinity for noncovalent dimerization, while the so-called enhanced variant (Fc-eYFP) lacked these dimerization-promoting mutations [17]. In addition we made use of the fact that expressed chemical ligation leads to the formation of a cysteine residue (Fig.…”

“…The cysteine residues of the Fc-YFP constructs, are situated in such a way that they are amenable to the formation of covalent disulfide crosslinks via oxidation [17]. In a control variant (Fc-bYFP), the cysteine residues of the monovalent constructs were blocked by reaction with N-methylmaleimide [17]. The different Fc-YFP variants were assembled on SAMs of bCD [17].…”

Supramolecular Protein Immobilization on Lipid Bilayers

“…By incorporation of theses ynthetic receptors on as olids upport, proteins containing the correspondingl igand can be immobilized. [20,26,[31][32][33][34][35][36][37][38][39] There are, however, only very limited examples of the use of supramolecular chemistry fort he immobilization of proteins on lipid bilayers. The potentialo f as upramolecular approach forp rotein immobilization on lipid membranes is most impressively illustrated by the membrane incorporation of nitrilotriacetic acids that recruit His 6 -tagged proteins upon addition of nickel.…”