Insight into the complexation mode of bis(nitrilotriacetic acid) (NTA) ligands with Ni(2+) involved in the labeling of histidine-tagged proteins.
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.
Brellier
Marie
M
Université de Strasbourg, Faculté de Pharmacie CNRS/UMR 7199, 74, route du Rhin, 67401 Illkirch, France. baati@bioorga.u-strasbg.fr
Barlaam
Bernard
B
Mioskowski
Charles
C
Baati
Rachid
R
D002151:Calorimetry; D005454:Fluorescence Polarization; D006639:Histidine / Q000378:metabolism*; D008024:Ligands; D009532:Nickel / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry* / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013194:Staining and Labeling
D008024:Ligands; D009942:Organometallic Compounds; D011506:Proteins; D006639:Histidine; D009532:Nickel; D009571:Nitrilotriacetic Acid