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Synthesis and copper(II)-binding properties of the N-terminal peptide of human alpha-fetoprotein.

ABSTRACT: The N-terminal native sequence tripeptide of alpha-fetoprotein, L-threonyl-L-leucyl-L-histidine N-methylamide, was synthesized and its interaction with Cu(II) ions was investigated by potentiometric titration at 25 degrees C in 0.15 M-NaCl and by visible-absorption, e.p.r. and n.m.r. spectroscopy. Analyses of the results in the pH range 4-10 indicated the presence of multiple complex species in solution: MHL, MH-2L, MHL2, ML2 and MH-1L2, where M, H and L represent metal ion, proton and ligand anion respectively. Only the species MH-2L and MH-1L2 are present in significant amounts at physiological pH. The results of the visible-absorption spectroscopy are consistent with the findings of species distribution that MH-2L is the major complex species detected above physiological pH that has the spectral characteristics of lambda max. = 523 nm and epsilon max. = 98 M-1.cm-1. The nine superhyperfine lines in e.p.r. spectra of the major species MH-2L strongly support the co-ordination of four nitrogen atoms by Cu(II). Both 1H- and 13C-n.m.r. studies suggest that the species MH-2L is a square-planar complex. The results from the equilibrium-dialysis experiments showed that this peptide is able to compete with albumin for Cu(II) ions. At equimolar concentrations of albumin and the peptide, about 52% of the Cu(II) was bound to the peptide. The possibility that alpha-fetoprotein plays an important role as the Cu(II)-transport protein in fetal life is discussed.

SUBMITTER: Lau SJ

PROVIDER: S-EPMC1135651 | biostudies-other | 1989 Feb

REPOSITORIES: biostudies-other

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Project description:Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a ??? protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional ??? folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2/??? zinc finger domain, the C-terminal finger of the Wilms' tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)-WT1-4 followed by fluorescence spectroscopy yielded the effective pKa(eff) values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKa(eff) values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)-WT1-4, Kf(ML) value of 7.5 × 10(12) M(-1), with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least -17.6 kcal/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)-WT1-4 to those from the model peptide Zn(II)-GGG-Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent Kf(ML) values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc finger proteins and support the conclusion that the cost of protein folding in most zinc finger proteins is ?+4.2 kcal/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, -17.6 kcal/mol.

Project description:The reactions of CuX2 (X = Cl, Br) with dipinodiazafluorenes yielded four new complexes [CuX2L1]2 (X = Cl (1), Br (2), L1 = (1R,3R,8R,10R)-2,2,9,9-Tetramethyl-3,4,7,8,9,10-hexahydro-1H-1,3:8,10-dimethanocyclopenta [1,2-b:5,4-b']diquinolin-12(2H)-one) and [(CuX2)2L2]n (X = Cl (3), Br (4), L2 = (1R,3R,8R,10R,1'R,3'R,8'R,10'R)-2,2,2',2',9,9,9',9'-Octamethyl-1,1',2,2',3,3',4,4',7,7',8,8',9,9',10,10'-hexadecahydro-1,3:1',3':8,10:8',10'-tetramethano-12,12'-bi(cyclopenta [1,2-b:5,4-b']diquinolinylidene). The complexes were characterized by IR and EPR spectroscopy, HR-ESI-MS and elemental analysis. The crystal structures of compounds 1, 2 and 4 were determined by X-ray diffraction (XRD) analysis. Complexes 1-2 have a monomeric structure, while complex 4 has a polymeric structure due to additional coordinating N,N sites in L2. All complexes contain a binuclear fragment {Cu2(μ-X)2×2} (X = Cl, Br) in their structures. Each copper atom has a distorted square-pyramidal coordination environment formed by two nitrogen atoms and three halogen atoms. The Cu-Nax distance is elongated compared to Cu-Neq. The EPR spectra of compounds 1-4 in CH3CN confirm their paramagnetic nature due to the d9 electronic configuration of the copper(II) ion. The magnetic properties of all compounds were studied by the method of static magnetic susceptibility. For complexes 1 and 2, the effective magnetic moments are µeff ≈ 1.87 and 1.83 µB (per each Cu2+ ion), respectively, in the temperature range 50-300 K, which are close to the theoretical spin value (1.73 µB). Ferromagnetic exchange interactions between Cu(II) ions inside {Cu2(μ-X)2X2} (X = Cl, Br) dimers (J/kB ≈ 25 and 31 K for 1 and 2, respectively) or between dimers (θ' ≈ 0.30 and 0.47 K for 1 and 2, respectively) were found at low temperatures. For compounds 3 and 4, the magnetic susceptibility is well described by the Curie-Weiss law in the temperature range 1.77-300 K with µeff ≈ 1.72 and 1.70 µB for 3 and 4, respectively, and weak antiferromagnetic interactions (θ ≈ -0.4 K for 3 and -0.65 K for 4). Complexes 1-4 exhibit high catalytic activity in the oxidation of alkanes and alcohols with peroxides. The maximum yield of cyclohexane oxidation products reached 50% (complex 3). Based on the data on the study of regio- and bond-selectivity, it was concluded that hydroxyl radicals play a decisive role in the oxidation reaction. The initial products in reactions with alkanes are alkyl hydroperoxides.

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Synthesis and copper(II)-binding properties of the N-terminal peptide of human alpha-fetoprotein.

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