Separation Of Recombinant Proteins Research Articles

Thermochemical and conformational studies of Ni(II) and Zn(II) ternary complexes of alternative metal binding peptides with nitrilotriacetic acid

Energy-resolved threshold collision-induced dissociation (TCID) of negatively-charged [amb5-H+M(II)+NTA-2H]− ternary complexes were studied for how they competitively dissociated into the products [amb5-3H+M(II)]− + NTA or [NTA-3H+M(II)]− + amb5, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contained one of the alternative metal binding-5 (amb5) heptapeptides with primary structures acetyl-Aa1-Aa2-Gly3-Pro4-Tyr5-Aa6-Aa7, where the amino acids Aa1,2,6,7 included the potential metal binding sites of Asp, His, and/or Cys. The study included heptahistidine which represents the His affinity tag commonly used in immobilized metal affinity chromatography (IMAC) separation of recombinant proteins. Molecular modeling using PM6 located low energy, geometry-optimized conformers of the ternary complexes and their products which had collision cross sections that agreed with those measured by ion mobility. Using the molecular parameters, including vibrational and rotational frequencies, of the reactant and products from the molecular modeling, the energy-dependent intensities of the two product channels were modeled using a competitive TCID method to determine their threshold energies which equated to the two product channels 0 K dissociation enthalpies (ΔH0). Statistical mechanics thermal corrections provided the 298 K enthalpies (ΔH298) and Gibbs free energies (ΔG298) for the association reactions [amb5-3H+M(II)]− + NTA ↔ [amb5-H+M(II)+NTA-2H]− and [NTA-3H+M(II)]− + amb5 ↔ [amb5-H+M(II)+NTA-2H]−. These results were compared to determine which amb5 were effective as alternative tags for IMAC purification of recombinant proteins and is a routine that can be generally applied to compare the thermochemistry of metal ion ternary complexes.