a-study-complexation-mercury-ii-with-dicysteinyl-tetrapeptides

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

Winston-Salem State University

The conformation of the non-glycosylated recombinant form of the extracellar domain of rat MOG (rMOG(1-125)) dissolved in different solvent conditions was studied by CD spectroscopy. The results show that rMOG(1-125) exhibits a predominantly beta sheet conformation in aqueous buffer solution at pH 7.5 and that this 'beta-form' is stabilized by zwitterionic phospholipids, DPC and LPCP. The alpha helical content of the protein can increase from 9% to up to 20% when TFE or anionic detergent LPAP and SDS are added.

Extracellular domain of myelin oligodendrocyte glycoprotein (MOG) exhibits solvent-dependent conformational transitions.

Selective high-affinity ligands (SHALs) were selected as substitutes for monoclonal antibodies (mAbs) to deliver radioisotopes to malignant tumors. Because a SHAL (5 KD) is considerably smaller in comparison to an antibody (150 KD), a significant therapeutic index (TI) enhancement for radioimmunotherapy (RIT) is anticipated. The antibody-antigen (Ab-Ag) model system chosen for the development of SHALs consists of Lym-1, a MAb with proven selectivity in non-Hodgkin's lymphoma (NHL) patients and its well-characterized Ag, the beta subunit of HLA DR10. Whereas Lym-1 is readily available, the subunit of HLA-DR10 is not. Native, heterodimeric (alpha and beta subunits) HLA-DR10 can be purified from Raji cells, which are known to overexpress this Ag. Inconsistent homogeneity between preparations of HLA-DR10 solubilized in the presence of detergents prompted us to express a recombinant form of the beta subunit of HLA-DR10 in Escherichia coli. Negligible production yields (<or=50 microg/L) were achieved by the expression of the full-length protein in a soluble form. By contrast, yields of 240 mg/L were obtained by expressing only the extracellular domain (ED) of the beta subunit of HLA-DR10 in an insoluble form (inclusion bodies). The recovery yield of refolded protein was 75%. Circular dichroism (CD) and Lym-1 binding studies indicated that the recombinant ED of the beta subunit of HLA-DR10 was properly folded. Therefore, this recombinant protein can be used as a surrogate for native heterodimeric HLA DR10 for the in vitro selection of SHALs and related targeting molecules.

Recombinant expression of the beta-subunit of HLA-DR10 for the selection of novel lymphoma targeting molecules.

The purpose of this work is to characterize the interactions of cyclooctapeptides (CP) containing glutamyl and/or cysteinyl residues with common heavy-metal ions in order to facilitate the design of cyclopeptides as sensors for metal ions. Isothermal titration calorimetry studies show that cyclooctapeptides containing glutamyl and/or cysteinyl residues bind these Hg(2+) and Pb(2+) over Cd(2+) and other common metal ions. Differential binding isotherms, in their interactions with Hg(2+), support a two-binding site model, whereas pertinent interactions with Pb(2+) support a 2:1 stoichiometry, suggesting a CP/Pb(2+)/CP mode of complexation. The cyclooctapeptide containing both glutamyl and cysteinyl residues shows a significant binding affinity for Hg(2+) (K(a)=7.6x10(7)M(-1)), which is both enthalpically and entropically driven. The fluorescence of these cyclooctapeptides showed pronounced fluorescence quenching responses to Hg(2+) over Pd(2+) and Cd(2+). Stern-Volmer analyses of the dependence of fluorescence intensity on Hg(2+) and Pb(2+) are reported. The observed trends are useful for the design of Hg(2+) sensors based on fluorophore-tagged cyclooctapeptides.

Thermodynamics and fluorescence studies of the interactions of cyclooctapeptides with Hg2+, Pb2+, and Cd2+.

Fluorogenic cyclooctapeptides and metal ion sensing.

The crystal structure of the title compound, C(12)H(11)NS, features parallel chains of alternating N-H⋯S hydrogen-bonded mirror-image conformers along [10[Formula: see text]]. The mol-ecular conformation is that of an envelope, with all of the framework atoms except one close to a mean plane (rms deviation 0.054 Å); one C atom of the cyclo-hexene-thione ring forms the envelope flap, which makes a dihedral angle of 48.6 (1)° with the rest of the mol-ecule. There is a π-π* inter-action between pairs of enanti-omers in adjacent chains; the distance between parallel planes is 3.466 (1) Å.

1,2-Dihydro-9H-carbazole-4(3H)-thione.

The present study was undertaken to gain insight into the associations of mercury(II) with dicysteinyl tripeptides in buffered media at pH 7.4. We investigated the effects of increasing the distance between cysteinyl residues on mercury(II) associations and complex formations. The peptide-mercury(II) formation constants and their associated thermodynamic parameters in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered solutions were evaluated by isothermal titration calorimetry. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides in ammonium formate buffered solutions were characterized by LTQ Orbitrap mass spectrometry. The results from these studies show that n-alkyl dicysteinyl peptides (CP 1-4), and an aryl dicysteinyl peptide (CP 5) can serve as effective "double anchors" to accommodate the coordination sites of mercury(II) to form predominantly one-to-one Hg(peptide) complexes. The aryl dicysteinyl peptide (CP 5) also forms the two-to-two Hg(2)(peptide)(2) complex. In the presence of excess peptide, Hg(peptide)(2) complexes are also detected. Notably, increasing the distance between the ligating groups or "anchor points" in CP 1-5 does not significantly affect their affinity for mercury(II). However, the enthalpy change (ΔH) values (ΔH(1) ~ -91 kJ mol(-1) and ΔH(2) ~ -66 kJ mol(-1)) for complex formation between CP 4 and 5 with mercury(II) are about one and a half times larger than the related values for CP 1, 2 and 3 (ΔH(1) ~ -66 kJ mol(-1) and ΔH(2) ~ 46 kJ mol(-1)). The corresponding entropy change (ΔS) values (ΔS(1) ~ -129 J K(-1) mol(-1) and ΔS(2) ~ -116 J K(-1) mol(-1)) of the structurally larger dicysteinyl peptides CP 4 and 5 are less entropically favorable than for CP 1, 2 and 3 (ΔS(1) ~ -48 J K(-1) mol(-1) and ΔS(2) ~ -44 J K(-1) mol(-1)). Generally, these associations result in a decrease in entropy, indicating that these peptide-mercury complexes potentially form highly ordered structures. The results from this study show that dicysteinyl tripeptides are effective in binding mercury(II) and they are promising motifs for the design of multi-cysteinyl peptides for binding more than one mercury(II) ion per peptide.

Evaluation of the association of mercury(II) with some dicysteinyl tripeptides.

In order to gain more insight into the associations of mercury(II) with cysteinyl peptides, we investigated the effect of increasing cysteinyl residues on complex type formations. Three series of di-, tri-, and tetra-cysteinyl peptides, D[CGD]nCG (CP 2A, CP 3A, and CP 4A), E[CEG]nCG (CP 2B, CP 3B, and CP 4B) and E[CDG]nCG (CP 2C, CP 3C, and CP 4C), where n=1, 2, or 3, were prepared by microwave-assisted solid phase peptide synthesis. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides were characterized by electrospray orbitrap mass spectrometry utilizing complex specific mercury isotopic patterns. In equimolar mercury(II) to peptide ratio, all three series of di-, tri-, and tetra-cysteinyl peptides form predominantly the 1:1Hg(peptide) complex type, indicating that the intervening amino acid residues do not elicit preferential complex type formation. However, in non-equivalent mercury(II) to peptide ratio, the number of cysteinyl residues has a significant effect on the Hg:peptide stoichiometry in the complex formed. For example, in four times excess peptide, the 1:2Hg(peptide)2 and 1:1Hg(peptide) complexes are formed for di-cysteinyl peptides but not for the tri- and tetra-cysteinyl peptides. In contrast, the 2:1Hg2(peptide) and 1:1Hg(peptide) complexes are formed for the tri- and tetra-cysteinyl peptides. In excess mercury(II), CP 4C formed exclusively the 2:1Hg2(peptide) complex. The exact number of deprotonations observed for each complex could be derived from its signature mercury isotope pattern and monoisotopic peak mass. These multi-cysteinyl peptides present an attractive option for mercury chelation or environmental heavy metal remediation.

Synthesis and ESI mass spectrometric analysis of the association of mercury(II) with multi-cysteinyl peptides.

The purpose of this study is to understand the interactions of some antibacterial cationic amphipathic cyclooctapeptides with calcium(II) and their secondary structural preferences. The thermodynamic parameters associated with calcium(II) interactions, between the antibacterial active cyclooctapeptides (COP 1-6) and those that did not exhibit significant activities (COP 7-9), were studied by isothermal titration calorimetry. Calcium(II) binding in the absence and presence of micellar dodecylphosphocholine (DPC), a membrane mimicking detergent, was conducted by circular dichroism (CD). Both groups of cyclopeptides showed weak binding affinities for calcium(II) (Kb ca. 10(-3) M(-1)). However, CD data showed that the antimicrobial peptides COP 1-6 adopted a twisted beta-sheet structure (positive CD absorption band at ca. 203 nm) in the presence of calcium(II) in micellar DPC. In contrast, COP 7-9, which lacked antibacterial activity, adopted a different conformational structure (negative CD absorption band at ca. 203 nm). These results indicate that these cyclopeptides could adopt secondary structural preferences in the presence of calcium(II) amidst a hydrophobic environment to elicit their antibacterial activity. These findings could be useful in facilitating the design of cyclopeptide derivatives that can adopt this beta-sheet-like secondary structure and, thereby, provide a useful molecular template for crafting antibacterial compounds.

Article	Year
Extracellular domain of myelin oligodendrocyte glycoprotein (MOG) exhibits solvent-dependent conformational transitions. Protein and peptide letters\| PubMed ID: 14561137	2003
Recombinant expression of the beta-subunit of HLA-DR10 for the selection of novel lymphoma targeting molecules. Cancer biotherapy & radiopharmaceuticals\| PubMed ID: 17803448	2007
Thermodynamics and fluorescence studies of the interactions of cyclooctapeptides with Hg2+, Pb2+, and Cd2+. Bioorganic & medicinal chemistry\| PubMed ID: 18396049	2008
Fluorogenic cyclooctapeptides and metal ion sensing. Advances in experimental medicine and biology\| PubMed ID: 19400106	2009
1,2-Dihydro-9H-carbazole-4(3H)-thione. Acta crystallographica. Section E, Structure reports online\| PubMed ID: 21754054	2011
Evaluation of the association of mercury(II) with some dicysteinyl tripeptides. Bioorganic chemistry\| PubMed ID: 22784828	2012
Synthesis and ESI mass spectrometric analysis of the association of mercury(II) with multi-cysteinyl peptides. Journal of inorganic biochemistry\| PubMed ID: 24413258	2014
Secondary Structural Preferences of Some Antibacterial Cyclooctapeptides in the Presence of Calcium(II). International journal of medicinal chemistry\| PubMed ID: 25379288	2012

Maria Ngu-Schwemlein

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