Super‐Chelators for Advanced Protein Labeling in Living Cells

Abstract

Live-cell labeling, super-resolution microscopy, single-molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N-nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA-based small interaction pairs described so far. Coupled to bright organic fluorophores with fine-tuned photophysical properties, the super-chelator probes were delivered into human cells by chemically gated nanopores. These super-chelators permit kinetic profiling, multiplexed labeling of His - and His -tagged proteins as well as single-molecule-based super-resolution imaging.

Multivalent Chelators for In Vivo Protein Labeling

“…[17] In contrast to the more common nickel-mediated interaction, Co 3+ complexes between the Histagged protein and the NTAr esulted in ah igh thermodynamic and kinetic stability of the complex with extended longterm stability (t 1/2 % 7days). [19] The1 00-fold higher dissociation constant of hexaNTAc ompared to trisNTAa llowed ak inetic discrimination between both MCHs in the presence of the competitor imidazole.B ased on this, trisNTAb ound to His 6 -tagged proteins dissociated within minutes in the presence of imidazole,w hereas the hexaNTA-Alexa Fluor 647 showed kinetically stable and persistent binding to His 12 -tagged proteins.C onversely,t he tunable and distinct kinetic profile of hexaNTAcompared to trisNTAe nabled the labeling of His 12 -tagged proteins with hexaNTAprior to the decoration of His 6 -tagged proteins with trisNTA. Theo xidation of Co 2+ to Co 3+ was also explored to decorate liposomes with His-tagged proteins as well as antigens.H ere,c obalt chelated in porphyrin-phospholipids was embedded in al ipid bilayer and allowed the stable capturing of His-tagged conjugates via non-covalent interactions,r esulting in an essentially irreversible anchoring to liposomes.…”

“…[19] Conventional fluorescence microscopy usually allows multiplexing owing to orthogonal excitation and emission spectra of different fluorophores.H owever,t he kinetic discrimination of different His-tags by hexaNTAa nd trisNTAe nabled the precise localization of two target POIs based on probes coupled to the same fluorophore.T heir tunable kinetics and distinct dissociation profiles provided as imple and generic approach for the use of as ingle fluorophore to image different targets.K inetic profiling was utilized in single-molecule-based super-resolution imaging and is,i np rinciple,a ble to surpass the limitations of multicolor microscopy. [19] Conventional fluorescence microscopy usually allows multiplexing owing to orthogonal excitation and emission spectra of different fluorophores.H owever,t he kinetic discrimination of different His-tags by hexaNTAa nd trisNTAe nabled the precise localization of two target POIs based on probes coupled to the same fluorophore.T heir tunable kinetics and distinct dissociation profiles provided as imple and generic approach for the use of as ingle fluorophore to image different targets.K inetic profiling was utilized in single-molecule-based super-resolution imaging and is,i np rinciple,a ble to surpass the limitations of multicolor microscopy.…”

“…Thes pecificity of trisNTAi sh ighlighted by the fact that MeCP2 harbors an endogenous sequence of seven consecutive histidines in an unstructured C-terminal region. [19] This approach permitted instantaneous imaging and lead to live-cell super-resolution imaging of the nuclear lamina by dSTORM. [53] Here,t he MCH-induced proximity was used to covalently attach the probe via ac ysteine-specific a-chloroacetamide group,a n approach analogous to that already established for proteins at the plasma membrane.…”

“…Exploiting a molecular avidity effect, tris NTA was recently advanced to distinguish between His 12 ‐tagged and His 6 ‐tagged proteins. By linking two cyclic tris NTAs via a short peptidic linker, a new class of hexavalent NTA ( hexa NTA, Figure c) chelators was defined, exhibiting subnanomolar affinities and extremely stable protein labeling even in the presence of 30 m m imidazole ( k off ≈10 −5 s −1 ) . The 100‐fold higher dissociation constant of hexa NTA compared to tris NTA allowed a kinetic discrimination between both MCHs in the presence of the competitor imidazole.…”

“…Recently, the hexa NTA was utilized in combination with tris NTA for the multiplexed labeling of His 6 ‐ and His 12 ‐tagged proteins . Conventional fluorescence microscopy usually allows multiplexing owing to orthogonal excitation and emission spectra of different fluorophores.…”