Immobilization of Ferrocene-Modified SNAP-Fusion Proteins

Abstract

The supramolecular assembly of proteins on surfaces has been investigated via the site-selective incorporation of a supramolecular moiety on proteins. To this end, fluorescent proteins have been site-selectively labeled with ferrocenes, as supramolecular guest moieties, via SNAP-tag technology. The assembly of guest-functionalized SNAP-fusion proteins on cyclodextrin- and cucurbit [7]uril-coated surfaces yielded stable monolayers. The binding of all ferrocene fusion proteins is specific as determined by surface plasmon resonance. Micropatterns of the fusion proteins, on patterned cyclodextrin and cucurbituril surfaces, have been visualized using fluorescence microscopy. The SNAP-fusion proteins were also immobilized on cyclodextrin vesicles. The supramolecular SNAP-tag labeling of proteins, thus, allows for the assembly of modified proteins via supramolecular host-guest interaction on different surfaces in a controlled manner. These findings extend the toolbox of fabricating supramolecular protein patterns on surfaces taking advantage of the high labeling efficiency of the SNAP-tag with versatile supramolecular moieties.

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

“…In the case of the ferrocene, ferrocene-substituted benzylguanine was covalently attached to a cysteine residue within the guanine binding pocket of the SNAP-tag (Fig. 4) [15]. For conceptual studies, we used fluorescent proteins for their robustness and intrinsic fluorescence making them ideal bio-probes for the use of fluorescence spectroscopy and microscopy.…”

“…For single, site-specific labelling we employed SNAP-tag technology, which is a genetically encoded protein tag, to efficiently label proteins with guest moieties featuring an O 6 -benzylguanine moiety such as adamantane [14] and redoxactive ferrocene [15]. In the case of the ferrocene, ferrocene-substituted benzylguanine was covalently attached to a cysteine residue within the guanine binding pocket of the SNAP-tag (Fig.…”

Stimuli‐Responsive Cucurbit[n]uril‐Mediated Host‐Guest Complexes on Surfaces

“…[67] We have reported successful ways to anchor proteins, viruses, bacteria and cells using CB[n]-mediated complexation on SAMs. [73,74,[88][89][90][91][92][93][94][95][96][97] A focus point has been to employ siteselectively guest-labeled proteins, which can be used for oriented positioning of proteins, as for example demonstrated on CB [7] SAMs. Mono-and divalent ferrocenylated yellow fluorescent protein were prepared and these formed stable inclusion complexes on CB [7] SAMs (method Figure 5b).…”

“…Mono-and divalent ferrocenylated yellow fluorescent protein were prepared and these formed stable inclusion complexes on CB [7] SAMs (method Figure 5b). [88,89] We designed several dynamic supramolecular CB [7] surfaces that are suitable for adhesion of cells. [88,90] Ferrocenylatedmodified integrin-binding Arg-Gly-Asp (RGD) peptides were anchored to CB [7] SAMs.…”

Engineered SNAP-MBD2b proteins for specific recognition of methylated DNA

“…It is an engineered variant based on human repair protein O 6 -alkylguanine-DNA-alkyltransferase (hAGT) that covalently reacts with O 6 -benzyl guanine (BG) derivatives [16,17]. SNAP-tag technique has been described not only for the study of the interactions between proteins, immobilization on solid surface, and pull-down assays, but also for the subcellular localizing such as the nucleus, endoplasmic reticulum [17][18][19][20][21].…”