Insight
into the Coordination and the Binding Sites
of Cu<sup>2+</sup> by the Histidyl-6-Tag using Experimental and Computational
Tools
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Abstract
His-tags
are specific sequences containing six to nine subsequent histydyl
residues, and they are used for purification of recombinant proteins
by use of IMAC chromatography. Such polyhistydyl tags, often used
in molecular biology, can be also found in nature. Proteins containing
histidine-rich domains play a critical role in many life functions
in both prokaryote and eukaryote organisms. Binding mode and the thermodynamic
properties of the system depend on the specific metal ion and the
histidine sequence. Despite the wide application of the His-tag for
purification of proteins, little is known about the properties of
metal-binding to such tag domains. This inspired us to undertake detailed
studies on the coordination of Cu<sup>2+</sup> ion to hexa-His-tag.
Experiments were performed using the potentiometric, UV–visible,
CD, and EPR techniques. In addition, molecular dynamics (MD) simulations
and density functional theory (DFT) calculations were applied. The
experimental studies have shown that the Cu<sup>2+</sup> ion binds
most likely to two imidazoles and one, two, or three amide nitrogens,
depending on the pH. The structures and stabilities of the complexes
for the Cu<sup>2+</sup>-Ac-(His)<sub>6</sub>-NH<sub>2</sub> system
using experimental and computational tools were established. Polymorphic
binding states are suggested, with a possibility of the formation
of α-helix structure induced by metal ion coordination. Metal
ion is bound to various pairs of imidazole moieties derived from the
tag with different efficiencies. The coordination sphere around the
metal ion is completed by molecules of water. Finally, the Cu<sup>2+</sup> binding by Ac-(His)<sub>6</sub>-NH<sub>2</sub> is much more
efficient compared to other multihistidine protein domains