2 research outputs found
Insight into the Coordination and the Binding Sites of Cu<sup>2+</sup> by the Histidyl-6-Tag using Experimental and Computational Tools
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
African Viper Poly-His Tag Peptide Fragment Efficiently Binds Metal Ions and Is Folded into an α‑Helical Structure
Snake
venoms are complex mixtures of toxic and often spectacularly biologically
active components. Some African vipers contain polyhistidine and polyglycine
peptides, which play a crucial role in the interaction with metal
ions during the inhibition of snake metalloproteases. Polyhistidine
peptide fragments, known as poly-His tags, play many important functions,
e.g., in metal ion transport in bacterial chaperon proteins. In this
paper, we report a detailed characterization of Cu<sup>2+</sup>, Ni<sup>2+</sup>, and Zn<sup>2+</sup> complexes with the EDDHHHHHHHHHG peptide
fragment (pHG) derived from the venom of the rough scale bush viper
(Atheris squamigera). In order to determine
the thermodynamic properties, stoichiometry, binding sites, and structures
of the metal–pHG complexes, we used a combination of experimental
techniques (potentiometric titrations, electrospray ionization mass
spectrometry, UV–vis spectroscopy, circular dichroism spectroscopy,
and electron paramagnetic resonance spectroscopy) and extensive computational
tools (molecular dynamics simulations and density functional theory
calculations). The results showed that pHG has a high affinity toward
metal ions. The numerous histidine residues located along this sequence
are efficient metal ion chelators with high affinities toward Cu<sup>2+</sup>, Ni<sup>2+</sup>, and Zn<sup>2+</sup> ions. The formation
of an α-helical structure induced by metal ion coordination
and the occurrence of polymorphic binding states were observed. It
is proposed that metal ions can “move along” the poly-His
tag, which serves as a metal ion transport pathway. The coordination
of Cu<sup>2+</sup>, Ni<sup>2+</sup>, and Zn<sup>2+</sup> ions to the
histidine tag is very effective in comparison with other histidine-rich
peptides. The stabilities of the metal–pHG complexes increase
in the order Zn<sup>2+</sup> < Ni<sup>2+</sup>≪ Cu<sup>2+</sup>