Cell Adhesion on RGD-Displaying Knottins with Varying Numbers of Tryptophan Amino Acids to Tune the Affinity for Assembly on Cucurbit[8]uril Surfaces

Abstract

Cell adhesion is studied on multivalent knottins, displaying RGD ligands with a high affinity for integrin receptors, that are assembled on CB[8]-methylviologen-modified surfaces. The multivalency in the knottins stems from the number of tryptophan amino acid moieties, between 0 and 4, that can form a heteroternary complex with cucurbit[8]uril (CB[8]) and surface-tethered methylviologen (MV2+). The binding affinity of the knottins with CB[8] and MV2+ surfaces was evaluated using surface plasmon resonance spectroscopy. Specific binding occurred, and the affinity increased with the valency of tryptophans on the knottin. Additionally, increased multilayer formation was observed, attributed to homoternary complex formation between tryptophan residues of different knottins and CB[8]. Thus, we were able to control the surface coverage of the knottins by valency and concentration. Cell experiments with mouse myoblast (C2C12) cells on the self-assembled knottin surfaces showed specific integrin recognition by the RGD-displaying knottins. Moreover, cells were observed to elongate more on the supramolecular knottin surfaces with a higher valency, and in addition, more pronounced focal adhesion formation was observed on the higher-valency knottin surfaces. We attribute this effect to the enhanced coverage and the enhanced affinity of the knottins in their interaction with the CB[8] surface. Collectively, these results are promising for the development of biomaterials including knottins via CB[8] ternary complexes for tunable interactions with cells.

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

“…[74] Also tryptophan (Trp) based protein immobilization was studied on this type of CB [8]-pseudorotaxane based SAMs. [91,95,97] Specificity and reversibility was typically verified in the experiments to confirm envisioned host-guest interactions to occur. In a recent study we prepared a focused set of integrin-targeting knottin variants with distinct numbers of tryptophans to vary affinities for the pseudorotaxane-based CB [8] SAMs.…”

“…[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).…”

“…In a recent study we prepared a focused set of integrin-targeting knottin variants with distinct numbers of tryptophans to vary affinities for the pseudorotaxane-based CB [8] SAMs. [91,95] The genetically engineered knottin constructs are able to simultaneously bind integrins and MV 2 + /CB [8] via one, two, three or four Trp heteroternary complexations. [95] Binding studies showed slightly higher binding affinities for knottins with a larger number of tryptophan residues while an increased extent of multilayer formation for the higher valent constructs, which we attributed to homoternary complex formation between tryptophans of different knottins and CB [8].…”

“…[91,95] The genetically engineered knottin constructs are able to simultaneously bind integrins and MV 2 + /CB [8] via one, two, three or four Trp heteroternary complexations. [95] Binding studies showed slightly higher binding affinities for knottins with a larger number of tryptophan residues while an increased extent of multilayer formation for the higher valent constructs, which we attributed to homoternary complex formation between tryptophans of different knottins and CB [8]. Tri-and tetravalent knottin constructs yielded the largest extent of adhered cell elongation and more pronounced focal adhesion formation.…”

“…Tri-and tetravalent knottin constructs yielded the largest extent of adhered cell elongation and more pronounced focal adhesion formation. [95] As an alternative to integrin-receptor mediated adhesion of cells on CB [8] SAMs, we have recently metabolically expressed naphthol moieties on the outer surface of non-adherent Jurkat cells (Figure 11c). [93] Specific host-guest was demonstrated on surfaces and the method potentially allows for programmable supramolecular interactions with spatial and temporal control over cell adhesion as well.…”