Supramolecular Control over Split‐Luciferase Complementation

Abstract

Supramolecular split‐enzyme complementation restores enzymatic activity and allows for on–off switching. Split‐luciferase fragment pairs were provided with an N‐terminal FGG sequence and screened for complementation through host‐guest binding to cucurbit[8]uril (Q8). Split‐luciferase heterocomplex formation was induced in a Q8 concentration dependent manner, resulting in a 20‐fold upregulation of luciferase activity. Supramolecular split‐luciferase complementation was fully reversible, as revealed by using two types of Q8 inhibitors. Competition studies with the weak‐binding FGG peptide revealed a 300‐fold enhanced stability for the formation of the ternary heterocomplex compared to binding of two of the same fragments to Q8. Stochiometric binding by the potent inhibitor memantine could be used for repeated cycling of luciferase activation and deactivation in conjunction with Q8, providing a versatile module for in vitro supramolecular signaling networks.

Synthetic mimics of biotin/(strept)avidin

“…39 Very recently, Brunsveld and co-workers used this homoternary complexation inside CB[8] to reconstitute a split luciferase. 40 Figure 11b shows schematic representations of two inactive fragments of firefly luciferase that had been appended with N -terminal FGG units (NFluc437 and CFluc398) which bind to each other with K d = 112 μM ( K a = 8,900 M −1 ). At concentrations 100-fold below K d , the NFluc437•CFluc398 assembly does not form significantly, and it was incompetent in the Promega luciferase assay.…”

“…(Reprinted by permission from refs 39 and 40. Copyright 2006 and 2016, American Chemical Society and Wiley-VCH).…”

A Binary Bivalent Supramolecular Assembly Platform Based on Cucurbit[8]uril and Dimeric Adapter Protein 14‐3‐3

“…In the absence of 14-3-3b ( Figure 3B,black line), high concentrations of the FGG-ERa peptide (IC 50 = 62 mm) were required to displace the AO,i nl ine with previous observations regarding the relatively weak competitive binding of FGG peptides. [16] In the presence of 14-3-3b,however, much lower concentrations of FGG-ERa sufficed to displace AO.Atintermediate 14-3-3b concentrations,amixed behavior reflecting the substoichiometric concentration of 14-3-3b as compared to Q8 and FGG-ERa became evident in the titration curves.I nt he presence of excess 14-3-3b,t he IC 50 value of the FGG-ERa peptide decreased to 2.5 mm,t hus representing a2 5-fold enhancement. Thet wo orthogonal assay types each address the concentration-dependent role of one of the two bivalent platforms,Q 8o r1 4-3-3b,a nd show that both assembly pathways (Figure 1) converge to the same end point through the double bivalency mechanism.…”

“…[3,[11][12][13] Notable examples include the dimerization of carbonic anhydrase by the use of as ynthetic foldamer platform [14,15] and the cucurbit [8]uril (Q8)-mediated functional reconstitution of as plit luciferase enzyme. [16] Thes tudy and modulation of protein-protein interactions (PPIs) is one of the most progressive areas of chemical biology and important for both fundamental research and drug discovery. [17,18] Protein assemblies frequently employ multivalent binding events,but the switching of this multivalency, for example between mono-and bivalent states,i sn ot readily modulated by classical small molecules.…”

Self-assembly of cucurbit[7]uril based triangular [4]molecular necklaces and their fluorescence properties

“…6a, 6b, 6e, 7 For example, CB[n] have been used to create chemical sensors, supramolecular materials, molecules and materials for drug solubilisation, delivery, and reversal, and as promotors of biological dimerisation events. 8 Over the past two decades, the strategic combination of rigid ligands and metal-ions with well defined coordination geometries has lead to the creation of a variety of functional self-assemblies. 9 Furthermore, some scientists have sought to extend the abilities of these systems by decorating them with molecular containers (e.g.…”