Figure 4.

Orb2A87 mutants H29A, H46A and H60A still bind to Ni²⁺ but only with half a binding site per monomer (A–C). ITC Ni²⁺ titration curves of Orb2A87 mutants, H29A (A); H46A (B); H60A (C). Both the heat compensation in the sample cell containing Orb2A87 as a function of time (top) and the resulting enthalpy changes as a function of molar addition of metal and the best fit to single binding site model (bottom) are shown; (D) H61Y does not bind to Ni-NTA resin. SDS-PAGE comparing the Ni-NTA pH 4.25 elution fraction of wild type (WT) Orb2A87 with Orb2A87 H61Y.