Tris-Nitrilotriacetic Acids of Subnanomolar Affinity Toward Hexahistidine Tagged Molecules

Abstract

Nitrilotriacetic acid (NTA) has moderate affinity (10 µM) for hexahistidine (His6) and is widely used in the purification of His6-tagged proteins. The affinity can be increased significantly (10 nM) through multivalency such as using a tris-NTA. We show that the binding affinity of tris-NTA is dependent on the flexibility and length of the spacer between the mono-NTA and the scaffold: the shorter the spacer, the higher the affinity. A series of biotinylated tris-NTA having different spacers were synthesized and used to prepare tris-NTA sensor chips for surface plasmon resonance measurement of binding affinity. Sub-nanomolar affinity can be achieved with a short spacer. The new high affinity tris-NTA enables the formation of stable complexes with hexahistidine containing molecules and provides a convenient method to non-covalently attach proteins to various surfaces.

Reversible Major Histocompatibility Complex I-Peptide Multimers Containing Ni2+-Nitrilotriacetic Acid Peptides and Histidine Tags Improve Analysis and Sorting of CD8+ T Cells

“…The present study demonstrates that reversible, highly effective MHC-peptide multimers can be prepared based on Ni 2ϩ -NTAHis tag chelate complexes. Previously branched tri-NTA complexes have been described that have up to subnanomolar K D for His 6 and His 10 tags (22)(23)(24). Our results demonstrate that highly stable chelate complexes can be formed with linear peptides carrying orthogonal NTA moieties; the binding of Ni 2ϩ -NTA 4 peptide (compound 4) to 2ϫHis 6 -tagged A2/Flu complexes with a K D of 19.6 pM is the highest affinity ever reported for NTA-His tag complexes ( Fig.…”

“…In compound 2, this distance is shorter than in compound 3. A previous study has shown that the avidity of branched tri-Ni 2ϩ -NTA molecules for His 6 -tagged molecules increased with the decreasing length of the spacer linking the NTA moieties to a central nitrogen (24). This argues that the NTA dimer compound 2 exhibited more avid binding than dimer compound 3, because of the shorter spacing distance xЈ.…”

Antibody Response to Polyhistidine-Tagged Peptide and Protein Antigens Attached to Liposomes via Lipid-Linked Nitrilotriacetic Acid in Mice

“…Thus, antibody responses to NTA-anchored antigens may be improved through further modifications of the NTA moiety to increase the affinity and stability of the NTA-polyhistidine interaction. Potential modifications include further increasing NTA valency (27), modifying the flexibility and length of the spacer between the mono-NTA and the scaffold (21), and incorporating hydrophobic shielding groups that impede removal of the chelated metal. These approaches are discussed in greater detail in recent publications from our laboratory (21,37).…”

“…To address this issue, we and others have developed facile synthetic routes to multivalent nitrilotriacetic acid adaptors with nanomolar affinities for polyhistidine-tagged proteins (K D [equilibrium dissociation constant], ϳ10 M and ϳ1 nM for monovalent and trivalent NTA [tris-NTA], respectively) ( Fig. 1) (3,21,22,27). This approach has shown promise for in vivo delivery; most notably, plasma membrane vesicles to which polyhistidinylated dendritic cell-targeting moieties and costimulatory molecules were engrafted via trivalent NTA elicited functional antitumor immunity upon administration to mice (44).…”

An Efficient Synthesis of 2,3-Diaminoacid Derivatives Using Phosphine Catalyst

“…2,3-Diaminoacids and their derivatives have attracted a great deal of attention due to their application as key structural fragments of biologically active compounds [1]- [7], and/or as ligands for metal complexes [8]- [13]. Various methods to prepare these compounds have been reported [1] [2] [4] [14]- [19].…”