The Cu­(II) and Zn­(II) binding abilities of Gly-His-Thr-Asp-amide (GHTD-am), a tetrapeptide coreleased from the pancreas along with insulin, were studied using UV–vis and circular dichroism spectroscopies, potentiometry, and calorimetry. GHTD-am is a very strong Cu­(II) chelator, forming a three-nitrogen complex with a conditional affinity constant C K at pH 7.4 of 4.5 × 1012 M–1. The fourth coordination site can be occupied by a solvent molecule or a ternary ligand, such as imidazole, with C K on the order of several hundred reciprocal molar. The Zn­(II) binding ability of GHTD-am is relatively weak, with C K values at pH 7.4 of 3.0 × 104 and 2.0 × 103 M–1 for the first and second GHTD-am molecule coordinated, respectively. These results are discussed in light of the modes of interactions of Zn­(II) and Cu­(II) ions with insulin. A direct effect of GHTD-am on the Zn­(II) interactions with insulin is unlikely, but its Cu­(II) complex may have a biological relevance because of its high affinity and ability to form ternary complexes.

The Aβ4−42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer’s Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aβ4−16 and Aβ4−9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aβ4−x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aβ4−16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aβ4−16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aβ4−16, Pd(II)-Aβ4−16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aβ complexes.

Neuroleptic drugs are widely applied in effective treatment of schizophrenia and related disorders. The lipophilic character of neuroleptics means that they tend to accumulate in the lipid membranes, impacting their functioning and processing. In this paper, the effect of four drugs, namely, thioridazine, olanzapine, sulpiride, and amisulpride, on neutral and negatively charged lipid bilayers was examined. The interaction of neuroleptics with lipids and the subsequent changes in the membrane physical properties was assessed using several complementary biophysical approaches (isothermal titration calorimetry, electron paramagnetic resonance spectroscopy, dynamic light scattering, and ζ potential measurements). We have determined the thermodynamic parameters, that is, the enthalpy of interaction and the binding constant, to describe the interactions of the investigated drugs with model membranes. Unlike thioridazine and olanzapine, which bind to both neutral and negatively charged membranes, amisulpride interacts with only the negatively charged one, while sulpiride does not bind to any of them. The mechanism of olanzapine and thioridazine insertion into the bilayer membrane cannot be described merely by a simple molecule partition between two different phases (the aqueous and the lipid phase). We have estimated the number of protons transferred in the course of drug binding to determine which of its forms, ionized or neutral, binds more strongly to the membrane. Finally, electron paramagnetic resonance results indicated that the drugs are localized near the water-membrane interface of the bilayer and presence of a negative charge promotes their burying deeper into the membrane.