Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd ≈ 6 μM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nɛ2 and Nδ1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP.

https://www.pnas.org/content/pnas/96/5/2042.full.pdf

Copper binding to the prion protein : structural implications of four identical cooperative binding sites

Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd ' 6 mM whereas a four-octarepeat peptide coopera- tively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordi- nation sphere of the copper is identical for 2, 3, or 4 octare- peats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the N«2 and Nd1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary require- ment for four octarepeats in the PrP.

Copper binding to the prion protein: Structural implications of four identical cooperative binding sites (octarepeat peptidesynuclear magnetic resonanceycircular dichroismyelectron spin resonance)

http://marcindyba.com/wp-content/uploads/publications/M_Dyba-Coord_Chem_Rev_1999_184_319-346.pdf

Specific structure–stability relations in metallopeptides

The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75 The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 70 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 122 (excitation lambda max = 476 nm; emission lambda max = 505 nm) With its peak position red-shifted 78 nm at pH 122, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 70 are also identical, suggesting that the chromophore binding site has returned to its native state Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure

Reversible Denaturation of Aequorea Green-Fluorescent Protein: Physical Separation and Characterization of the Renatured Protein

Publisher Summary This chapter discusses copper complexes that offer a physiological approach to the treatment of chronic diseases. Copper is recognized as an essential metalloelement just as sodium, potassium, magnesium, calcium, iron, zinc, chromium, vanadium, and manganese. Just as essential amino acids, essential fatty acids and essential cofactors (vitamins), essential metalloelements are required for normal metabolic processes but cannot be synthesized de novo, and daily dietary intake and absorption are required. The adult body contains between 1.4 mg (22 pmol) and 2.1 mg (33 pmol) of copper per kilogram of body weight while the infant body contains three times this amount, consistent with the fact that infant metabolic needs are that much greater than those of adults. The essentiality of copper is now understood as being based upon its recognized need for activation of copper-dependent enzymes. Complexed forms of copper also facilitate absorption, tissue distribution, and tissue utilization. In the nondisease state, these forms of copper account for the physiologic regulation of copper-dependent homeostatic processes. Consistent with the newly found role for copper in synthesis of neuroendocrine peptides, the copper complex of histidine has been found to increase the secretion of luteinizing hormone-releasing hormone (LH-RH) from granules of the median eminence.

Copper complexes offer a physiological approach to treatment of chronic diseases.

The plasma tripeptide glycyl-L-histidyl-L-lysine (GHL), when added at nanomolar concentrations to a wide group of cultured systems, produces a disparate set of responses ranging from the stimulation of growth and differentiation to outright toxicity1–3. Such diverse actions imply that this tripeptide mediates some basic biochemical function common to many types of cells and organisms. During the isolation of GHL we found the compound to co-isolate through a number of steps with approximately equimolar copper and about 1/5 molar iron1. Maximal effects on hepatoma cells (HTC4) were seen when the peptide was added with copper and iron to the growth medium1. Structure–function studies revealed that several tripeptides with a histidyl-lysyl linkage were nearly as active as GHL1. The association of GHL with copper and a homology similarity between the tripeptide and the copper transport sites on albumin and α-fetoprotein, where the cupric atom is bound to a histidyl residue adjacent to a basic residue, suggested that GHL may act as a copper transport factor4. We report here that the tripeptide readily forms complexes with copper(II) and enhances the uptake of the metal into cultured hepatoma cells.

Christopher M. Perkins

Papers

Growth-modulating plasma tripeptide may function by facilitating copper uptake into cells.

The structure of a copper complex of the growth factor glycyl-L-histidyl-L-lysine at 1.1 Å resolution

Synthesis, structures, and electrochemistry of copper(II)-mercaptide complexes

Electronic structure of iron complexes containing extended ligand .pi. systems

Electronic structure of iron complexes containing extended ligand .pi. systems