Insight into the Complexation Mode of Bis(nitrilotriacetic acid) (NTA) Ligands with Ni²⁺ Involved in the Labeling of Histidine‐Tagged Proteins

Abstract

According to literature reports and our own findings, the binding of new Ni(2+)-preloaded bis(nitrilotriacetic acid) (NTA) ligands with polyhistidine-tagged proteins has been found to be accompanied by a one- to two-order-of-magnitude increase in affinity, compared to the binding of a single Ni(2+)-preloaded NTA moiety. In spite of the introduction of a second NTA chelating group, a cooperative effect that is less than the theoretical maximum has been observed. Herein, we present a rational explanation for the observed stability of the ternary complex involving the postulated bis-NTA-(Ni(2+))(2) species and multivalent polyhistidine tags. We have found that prior to the formation of the ternary complex, the Ni(2+)-preloading step of bis-NTA ligands does not form the expected bis-NTA-(Ni(2+))(2) exclusively. Instead of the major formation of bis-NTA-(Ni(2+))(2) species, it appears that cyclic discrete 1:1 and 2:2 entities are predominantly formed. It is proposed that these species interact upon ring-opening with multivalent histidine tags. The occurrence of this phenomena accounts for the overall one- to two-order-of-magnitude increase in affinity of ternary complexes involving bis-NTA ligands.

Aptameric Peptide for One-Step Detection of Protein Kinase

“…Then the terminal carboxylate group of surface-confined DHLA was covalently conjugated with the free amino group of the NTA/Ni(II) complex, causing a frequency change of ∼−184 Hz (Figure S1B). The NTA/Ni(II) complex termination contains two free coordination sites occupied by water molecules that can be replaced by histidine residues, , so aptameric peptide IP 20 bearing a six-histidine tag could be immobilized specifically to obtain an IP 20 -functionalized QCM (shown as step 2). The immobilization process of IP 20 was monitored with the QCM in real time (Figure S1C).…”

Immobilization of bio-macromolecules on self-assembled monolayers: methods and sensor applications

“…68 Brellier et al reported that although bis-NTA binds with His 6 -tagged proteins via Ni 2+ with improved binding capabilities, however, a cooperative effect less than theoretical maximum was observed in this case. 69 It was suggested that the susceptibility of divalent molecules to form discrete species and oligomers is the reason for the absence of a strong cooperative effect. It was also observed that when bis-NTA ligands were preloaded with two equivalents of Ni 2+ , a number of discrete species with defined stoichiometries were generated instead of exclusive formation of bis-NTA-(Ni 2+ ) 2 species.…”

Biofunctional Surface Patterns Retaining Activity after Exposure to Whole Blood

“…Remarkably, the PLL- g -PEG biotin features from both single and binary patterns were still capable of coupling with streptavidin, even after 7 days in blood. This difference in the retention of bioactivity observed between the two moieties result from the difference in the affinity of protein/ligand interaction, where the biotin/streptavidin interaction possess a higher affinity per single ligand compared to the NTA-Ni 2+ /oligohistidine interaction ( K D ≈ 10 –14 and 10 –6 M for biotin/stretptavidin and NTA-Ni 2+ /oligohistidine, respectively), and the difference in the molecular architecture between PLL- g -PEG biotin and PLL- g -PEG NTA, where PLL- g -PEG biotin possesses a greater number of PEG side chains and fewer biotin groups compared to PLL- g -PEG NTA, where most of the PEG side chains are terminated with NTA groups (96%). The greater number of negatively charged NTA groups present on the surface leads to higher amount of nonspecific adsorption of positively charged proteins from blood .…”