Formation of Co(II), Ni(II), Zn(II) complexes of alternative metal binding heptapeptides and nitrilotriacetic acid: Discovering new potential affinity tags

Abstract

Using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) a new series of alternative metal binding-5 (amb5) heptapeptides that provide various combinations of His, Cys and Asp as potential metal binding sites are compared for their relative formation of complexes with the metal ions Co(II), Ni(II) and Zn(II). These amb5 peptides have the general primary structure acetyl-Aa1-Aa2-Gly3-Pro4-Tyr5-Aa6-Aa7, where Aa1,2,6,7 are the four amino acids that include the His, Cys, and/or Asp potential metal binding sites. The study includes two polyhistidine heptapeptides; the His-tag and His-tag with Pro4, because they represent the widely applied His affinity tag used in expression cloning and purification of recombinant proteins with immobilized metal affinity chromatography (IMAC). We compare these amb5 heptapeptides for their formation of the amb5+M(II) and amb5+M(II)+NTA complexes, where M = Co, Ni, or Zn, and NTA is nitrilotriacetic acid as a model for the binding of amb5 in a IMAC column. Overall, the results show that the inclusion of Asp at positions 1, 2, and 7; i.e., ac-Asp1-Asp2-Gly3-Pro4-Tyr5-Aa6-Asp7, generally favors the formation of amb5+Ni(II) and amb5+Ni(II)+NTA complexes, while the inclusion of Cys at positions 2 and 6; i.e., ac-Aa1-Cys2-Gly3-Pro4-Tyr5-Aa6-Cys7, with Aa1 = His, Cys or Asp, favors the formation of amb5+Zn(II) complexes. The comparison with the His-tag and competition between Ni(II) and Zn(II) for forming the amb5+M(II)+NTA complexes, showed the structure ac-Asp1-Asp2-Gly3-Pro4-Tyr5-His6-Aa7, with Aa7 = Cys or Asp, were the most selective for Ni(II) and forming the amb5+Ni(II)+NTA complex. Collision-induced dissociation of the single negatively-charged [amb5+Ni(II)+NTA]− shows it competitively dissociates into [amb5−3H+Ni(II)]− or [NTA−3H+Ni(II)]− while the doubly-charged [amb5+Ni(II)+NTA]2− complex dissociates into [NTA−3H+Ni(II)]− and [amb5J−H]−, indicating these complexes are realistic mimics for the complex inside an IMAC column.