A new, model-free calculation method to determine the coordination modes and distribution of copper(II) among the metal binding sites of multihistidine peptides using circular dichroism spectroscopy.
A new calculation method to determine microscopic protonation processes from CD spectra measured at different pH and Cu(II):ligand ratios was developed and used to give the relative binding strengths for the three histidines of hsPrP(84-114), a 31-mer polypeptide modeling the N-terminal copper(II) binding region of human (homo sapiens) prion protein. Mutants of hsPrP(84-114) with two or one histidyl residues have also been synthesized and their copper(II) complexes studied by CD spectroscopy. The 1-His models were analyzed first, and the molar CD spectra for the different coordination modes on the different histidines were calculated using the general computational program PSEQUAD. These spectra were deconvoluted into the sum of Gaussian curves and used as a first parameter set to calculate the molar spectra for the different coordination modes (3N and 4N coordination) and coordination positions (His85, His96 and His111) of the 2-His peptides. The calculation method therefore does not require the direct use of CD spectra measured in the smaller peptide models. This is a significant improvement over earlier calculation methods. In the same runs, the stepwise deprotonation pK(mic) values were refined and the pH-dependent distribution of copper(II) between the two histidines was determined. The results revealed the high, but different copper(II) binding affinities of the three separate histidines in the following order: His85 << His96His111. The calculation also showed that molar CD spectra which belong to the same coordination mode and coordination position in different ligands have very similar transition energies but different intensities. For this reason, direct transfer of molar CD spectra between different ligands may be a source of error, but the pK(mic) values and the copper(II) binding preferences are transferable from the 2-His peptides to the 3-His hsPrP(84-114).
Osz
Katalin
K
University of Debrecen, Department of Physical Chemistry, H-4010 Debrecen, Hungary.
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008432:Mathematical Computing; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011328:Prions / Q000737:chemistry*; D012984:Software; D013057:Spectrum Analysis
D010455:Peptides; D011328:Prions; D006639:Histidine; D003300:Copper