Strong and Reversible Monovalent Supramolecular Protein Immobilization

Abstract

Biological molecules such as proteins and oligonucleotides are the greatest source of inspiration for supramolecular chemists.[1] With the increased knowledge about supramolecular architectures in water, [2] applications of synthetic supramolecular architectures to investigate the biological molecules that initially inspired their design has become possible. The combination of synthetic supramolecular systems with proteins, for example, allows the control and study of these proteins.[3] The combination of designed supramolecular interactions with proteins allows for a powerful application of recognition motifs that are bio-orthogonal and thus provides an independent opportunity to modulate proteins. An important target in the field of protein nanotechnology, for example, is control over protein immobilization onto surfaces for diverse biochemical and biomedical applications.[4] Immobilization approaches based on biological supramolecular recognition motifs have been reported in recent years, such as DNA-protein interactions, [5] NiNTA-His 6 -tagged protein systems, [6] and the biotin and avidin interaction.[7] Supramolecular protein immobilization through synthetic supramolecular elements such as adamantane [8] and ferrocene [9] has also been reported; it relies on multivalent interactions of randomly and multiply attached supramolecules. However, a simple, strong, reversible, and monovalent supramolecular protein immobilization strategy is still required. A bio-orthogonal and monovalent supramolecularprotein system would allow for a high level of control over the homogeneity of the protein monolayers and site specificity of immobilization, whilst also displaying controllable binding kinetics.Herein we demonstrate how a single, synthetic supramolecular host-guest interaction allows for stable, but reversible, site-selective, monovalent protein immobilization on a self-assembled monolayer. This work utilizes the site-selective incorporation of ferrocene into a protein followed by immobilization onto a cucurbit [7]uril (CB[7]) monolayer. The technique allows printing of stable protein monolayers in well-defined formats to be achieved with controlled protein orientation and with subsequent replacement of the protein monolayer by a small synthetic ligand. The scope of this new immobilization strategy was investigated by using X-ray photoelectron (XPS) and Fourier transform IR reflection absorption (IR-RAS) spectroscopy, surface plasmon resonance (SPR), cyclic voltammetry (CV), atomic force (AFM) and fluorescence microscopy (FM).We used expressed protein ligation as a site-selective protein modification technique to insert a single supramolecular guest molecule.[10] This allows for the generation of a homogenously labeled protein and avoids random and multiple tagging of proteins, with concomitant problems in protein immobilization. The supramolecular guest (ferrocenylmethyl)trimethyl-ammonium iodide features a strong binding affinity of K % 10 11 m À1 to CB [7] in solution.[11] Consequently, we selected the protonata...

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.…”

“…[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. [90] These supramolecular RGD SAMs were used for adhering umbilical vein endothelial cells representing an early example of cell adhesion by the supramolecular ferrocene-CB [7] guest-host pair on gold surfaces.…”

Orthogonal Supramolecular Interaction Motifs for Functional Monolayer Architectures

“…[40] Their method does not require any prior modification or special treatment of CB[n] molecules, and is applicable for all members of the CB[n] family, at least CB [6][7][8] Jonkheijm and co-workers also used a CB [7] monolayer for the immobilization of the proteins through a monovalent supramolecular interaction. [41] Their technique allows printing of stable protein monolayers in well-defined formats to be achieved with controlled protein orientation and with subsequent replacement of the protein monolayer by a small synthetic ligand (Figure 2.15).…”

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.…”