The Scaffold Design of Trivalent Chelator Heads Dictates Affinity and Stability for Labeling His‐tagged Proteins in vitro and in Cells

Abstract

Small chemical/biological interaction pairs are at the forefront in tracing protein function and interaction at high signal-to-background ratios in cellular pathways. However, the optimal design of scaffold, linker, and chelator head still deserve systematic investigation to achieve the highest affinity and kinetic stability for in vitro and especially cellular applications. We report on a library of N-nitrilotriacetic acid (NTA)-based multivalent chelator heads (MCHs) built on linear, cyclic, and dendritic scaffolds and compare these with regard to their binding affinity and stability for the labeling of cellular His-tagged proteins. Furthermore, we describe a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we outline fundamental differences between the MCH scaffolds and define a cyclic trisNTA chelator that displays the highest affinity and kinetic stability of all reported reversible, low-molecular-weight interaction pairs.

Multivalent Chelators for In Vivo Protein Labeling

“…For MCHs in general and especially tris NTA, an intricate interplay between the molecular architecture of the scaffold and the MCH multivalency is apparent. As a result of our comprehensive study, a cyclic scaffold as well as a glutamate‐based linker is seen as optimal and superior for high‐affinity and stable binding of tris NTA to His‐tagged proteins . With regard to high‐accuracy protein labeling, key factors, including site‐selectivity and specificity, efficiency and rate of labeling, bioorthogonality to other labeling methods, close‐proximity as well as accessibility of the cognate partner, must all be considered.…”

“…Responsible for this are the drastic decrease of conformational freedom upon complex formation as well as the drop in flexibility of both interaction partners. Thus, the mobility of the linkers as well as the different MCH topologies (dendritic, linear, or cyclic) can severely affect the net free energy of MCH complex formation . Kinetically, the nanomolar K d values for MCH/His‐tag complexes are marked by very fast k on and extremely slow k off rates ( K d = k on / k off ; for example, tris NTA–His 6 ‐tag k on =1.6±0.4×10 5 m −1 s −1 and k off =0.34±0.01×10 −3 s −1 ) .…”

“…Most common for MCHs is the assembly of 2–4 NTAs on cyclic or dendritic scaffolds. A systematic investigation of the impact of tris NTA architectures revealed that cyclic tris NTAs grafted to a cyclam or cyclen scaffold in combination with a glutamate‐linked NTA are superior in terms of kinetic stability and affinity . In contrast, the linear as well as dendritic tris NTAs displayed a significantly lower kinetic stability compared to cyclic tris NTA as examined by surface plasmon resonance (SPR) spectroscopy.…”

“…Reproduced with permission from ref. . Copyright Wiley‐VCH, 2018. b) High‐affinity tris NTA–His‐tag interaction.…”

Simplified detection of polyhistidine-tagged proteins in gels and membranes using a UV-excitable dye and a multiple chelator head pair

“…Careful chemical and geometrical considerations have led to the assembly of the NTA moiety into higher-order structures, known as multivalent chelator heads (MCHs). The multivalent interactions of Ni 21 -MCHs (Nickel (II)-loaded MCH) increase the affinity toward His 6 -tag by a 1000-fold relative to Ni 21 -monoNTA (5)(6)(7)(8)(9). This optimization considerably increases both the applicability and popularity of His-Ni 21 -NTA-based approaches (10)(11)(12)(13)(14)(15).…”