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Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II) >> Cu(II) >> Ni(II) >= Zn(II) > Cd(II) similar to Co(II) > Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N-im-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.

Introduction Eukaryotic porins (or VDACs, Voltage Dependent Anion-selective Channels) are membrane proteins forming an aqueous pore channel in the mitochondrial outer membrane [1]. While bacterial porins structure has been determined by X-Ray diffraction, the structure of eukaryotic porins is still a matter of debate. Bacterial porins can “hole” the outer membrane by a peculiar structure called beta-barrel, since it is formed by 16 (on average) antiparallel amphipathic beta strands lining an aqueous environment [2,3]. Theoretical predictions largely agree in the indication of a predominant content of amphipathic beta strands in the eukaryotic porin structure. In addition to a very structured and compact beta-barrel, every prediction performed so far has highlighted in VDACc the presence of a segment of -helix, 19-20 amino acids long, with amphipatic features, at the N-terminal of the protein. The bacterial porins do not show such an helical structure and it is thus the most relevant addition to porins in eukaryotic cells. The N-terminal -helix was claimed to be part of the channel walls, to lie transiently upon one side of the phospholipid bilayer, to allow some motion to the protein during the voltage-gating or to interact specifically with other molecules like cytochrome C and the so called polyanion, a pore-activity inhibitor [4-6]. In this work we report about the physico-chemical characterization of the peptide corresponding to the human porin (VDAC1) sequence 1-19. The biological effects of the deletion of this segment are also shown. The results are discussed on the basis of the known functions of this intriguing membrane protein.

The prion protein (PrP(c)) is a copper-binding glycoprotein that can misfold into a beta-sheet-rich and pathogenic isoform (PrP(sc)) leading to prion diseases. The first non-mammalian PrP(c) was identified in chicken and it was found to keep many structural motifs present in mammalian PrP(c), despite the low sequence identity (approximately 40%) between the two primary structures. The present paper describes the synthesis and the coordination properties of some hexapeptide fragments (namely, PHNPGY , HNPGYP and NPGYPH) as well as a bishexapeptide (PHNPGYPHNPGY), which encompasses two hexarepeats. The copper(II) complexes were characterized by means of potentiometric, UV-vis, circular dichroism and electron paramagnetic resonance techniques. We also report the synthesis of three hexapeptides (PHNPGF, HNPGFP and NPGFPH), in which one tyrosine was replaced by phenylalanine as well as two bishexapeptides in which either one (PHNPGFPHNPGY and PHNPGYPHNPGF), or two tyrosines were replaced by phenylalanine, in order to check whether tyrosine was involved in copper(II) binding. Overall, the results indicate that the major copper(II) species formed by the chicken PrP dodecapeptides are stabler than the analogous species reported for the peptide fragments containing two octarepeat peptides from the mammalian prion protein. It is concluded that the presence of four prolyl residues, that are break points in copper coordination, induces the metal-assisted formation of macrochelates as well as the formation of binuclear species. Furthermore, it has been shown that the phenolic group is directly involved in the formation of copper binuclear species.

The synthesis and characterization of two β-cyclodextrins (β-CD) functionalized with two units of carnosine (β-alanyl- l -histidine) through the amino group, 6A,6C-(β-alanyl- l -histidine)-6A,6C-dideoxy-β-cyclodextrin (ACCDAH) and 6A,6D-(β-alanyl- l -histidine)-6A,6D-dideoxy-β-cyclodextrin (ADCDAH), are reported. NMR and C.D. data of the ligands indicate a different interaction of dipeptide chains with upper rim and cavity of β-CD. Analogously, spectroscopic and electrospray ionization mass spectrometry data show different copper(II) complex species formed by the two regioisomers. The ability of carnosine–cyclodextrin derivatives to bind copper ions in a head-to-tail fashion induces the formation of oligomeric species (up to hexamers) in the case of ACCDAH, where the two carnosine moieties are adjacent, while in the ADCDAH case the mutual interaction between the peptidic chains of two ADCDAH molecules allows the almost exclusive formation of a copper-assisted self-assembled dimeric species.

The systems that the 3-amino derivative of beta-cyclodextrin (CD3NH2) forms with the proton, the copper(II) ion and each of the enantionters of certain amino acids (alanine, phenylalanine, tyrosine and tryptophan) were investigated. The enantioselectivity shown by the potentiometric measurements carried out on the phenylalanine ternary systems was exploited in capillary electrophoresis by ligand exchange capillary electrophoresis (LECE) to obtain the separation of phenylalanine racemate. The tyrosine racemate was also separated by LECE. The comparison between thermodynamic and capillary electrophoresis (CE) results is discussed, in order to get a better insight into the separation mechanism.

The synthesis of epimers 6-deoxy-6-[N-tert-butoxycarbony1-β-alanyl-(S)histidylamino]-β-cyclodextrin and 6-deoxy-6-[N-tert-butoxycarbonyl-β-alanyl-(R)histidylamino]-β-cyclodextrin is reported. CD and NMR spectroscopy reveals that in aqueous solution these compounds are arranged in a ‘sleeping swan’ like conformation. H bonds between the NH of peptide bonds and the upper rim of CDs stabilize self-inclusion. The chirality of His influences the orientation of the self-included chain in the cavity, thus determining different properties for the two epimers.

Stability constant values and bonding details of the copper(II) complexes of the β-cyclodextrin functionalized with the carnosine dipeptide (β-alanyl-L-histidine) at its narrow (CDAH6) or at its wide (CDAH3) rim were determined in aqueous solution. The potentiometric and spectroscopic data (UV-vis, CD and EPR) show that the involvement of a secondary OH group induces drastic differences in the coordination properties of CDAH3, in comparison with those of CDAH6. Direct and indirect assays were carried out showing that the copper(II) complexes with the two cyclodextrin derivatives are SOD-mimics with high catalytic activity. In addition the complex species are scavenger compounds towards ˙OH radicals, giving rise to a particular kind of copper(II) complexes with a combined activity against two toxic radical species, O2˙− and ˙OH. The cyclodextrin moiety contributes to the scavenger activity, without damaging the cellular membranes of neuronal and red blood cells.