Diastereoselective DNA cleavage recognition by Ni(II) x Gly-Gly-His-derived metallopeptides

Site-selective DNA cleavage by diastereoisomers of Ni(II) x Gly-Gly-His-derived metallopeptides was investigated through high-resolution gel analyses and molecular dynamics simulations. Ni(II) x L-Arg-Gly-His and Ni(II) x D-Arg-Gly-His (and their respective Lys analogues) targeted A/T-rich regions; however, the L-isomers consistently modified a subset of available nucleotides within a given minor groove site, while the D-isomers differed in both their sites of preference and their ability to target individual nucleotides within some sites. In comparison, Ni(II) x L-Pro-Gly-His and Ni(II) x D-Pro-Gly-His were unable to exhibit a similar diastereoselectivity. Simulations of the above systems, along with Ni(II) x Gly-Gly-His, indicated that the stereochemistry of the amino-terminal amino acid produces either an isohelical metallopeptide that associates stably at individual DNA sites (L-Arg or L-Lys) or, with D-Arg and D-Lys, a noncomplementary metallopeptide structure that cannot fully employ its side chain nor amino-terminal amine as positional stabilizing moieties. In contrast, amino-terminal Pro-containing metallopeptides of either stereochemistry, lacking an extended side chain directed toward the minor groove, did not exhibit a similar diastereoselectivity. While the identity and stereochemistry of amino acids located in the amino-terminal peptide position influenced DNA cleavage, metallopeptide diastereoisomers containing L- and D-Arg (or Lys) within the second peptide position did not exhibit diastereoselective DNA cleavage patterns; simulations indicated that a positively charged amino acid in this location alters the interaction of the metallopeptide equatorial plane and the minor groove leading to an interaction similar to Ni(II) x Gly-Gly-His.     

Potential-modulated DNA cleavage by (N-salicylideneglycinato)copper(II) complex.

The interaction of aqua (N-salicylideneglycinato)copper(II) (Cu(salgly)2+) complex with calf thymus DNA has been investigated by cyclic voltammetry. Potential-modulated DNA cleavage in the presence of Cu(salgly)2+ complex was performed at a gold electrode in a thin layer cell. DNA can be efficiently cleaved by electrochemically reducing Cu(salgly)2+ complex to Cu(salgly)+ complex at -0.7 V (vs. Ag/AgCl). When the solution was aerated with a small flow of O2 during electrolysis, the extent of DNA cleavage was dramatically enhanced, and hydroxyl radical scavengers inhibited DNA cleavage. These results suggested that O2 and hydroxyl radical were involved in potential-modulated DNA cleavage reaction. The percentage of DNA cleavage was enhanced as the working potential was shifted to more negative values and the electrolysis time was increased. It was also dependent on the ratio of Cu(salgly)2+ complex to DNA concentration. The cleaved DNA fragments were separated by high performance liquid chromatography (HPLC). The experimental results indicated that the method for potential-modulated DNA cleavage by Cu(salgly)2+ complex was simple and efficient.     

Incorporation of β-Alanine in Cu(II) ATCUN Peptide Complexes Increases ROS Levels,DNA Cleavage and Antiproliferative Activity**

Redox-active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore leads to low ROS generation. In this work, we demonstrate that an exchange of the α-amino acid Gly with the β-amino acid β-Ala at position 2 (Gly2→β-Ala2) of the ATCUN motif reinstates ROS production (^•OH and H₂O₂). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these β-Ala2-containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for β-Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where β-Ala2 peptide complexes had lower IC₅₀ values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP-MS measurements.     

Thermal, spectral, magnetic and antimicrobial behaviour of new Ni(II), Cu(II) and Zn(II) complexes with a hexaazamacrocyclic ligand

New species of type MLCl₂·nH₂O (M:Ni, n = 6; M:Cu, n = 1.5 and M:Zn, n = 1; L: 1,8-bis(3′-ketopyridil)-1,3,6,8,10,13-hexaazacyclotetradecane, ligand resulted by 1,2-diaminoethane, nicotinamide and formaldehyde template condensation) were synthesised. The compounds were characterised by chemical analysis, ESI–MS, IR, NMR, UV–Vis-NIR and EPR spectroscopy as well as magnetic data at room temperature. The modifications in the IR and NMR spectra are in accordance with the condensation process. Electronic spectra indicate that Ni(II) adopts an octahedral stereochemistry while the surrounding of Cu(II) is square-pyramidal. The proposed stereochemistry was furthermore confirmed by magnetic moments and EPR spectrum at room temperature. The water is eliminated in one or two steps, respectively, while the oxidative degradation of the ligand and chloride decomposition occur in two steps. The final residues consist of the most stable metallic oxides as X-ray powder diffraction indicates. The newly synthesised compounds were evaluated for their antimicrobial effect against different bacterial and fungal strains.     

Synthesis, structure and comparison of the DNA cleavage ability of metal complexes M(II)L with the N-(2-ethoxyethanol)-bis(2-picolyl)amine ligand L (M = Co, Ni, Cu and Zn)

Complexes of a N,N-bis(2-picolyl)amine (bpa) derivative with a pendant ethoxyethanol side chain (bpa(CH2)2O(CH2)2OH) (1) with late divalent transition metal ions Co(II), Ni(II), Cu(II) and Zn(II) have been studied. All complexes, [[bpa(CH2)2O(CH2)2OH]Co(NO3)](NO3) (1Co), [[bpa(CH2)2O(CH2)2OH]Ni(NO3)](NO3) (1Ni), [[bpa(CH2)2O(CH2)2OH]Cu(H2O)(NO3)](NO3) (1Cu) and [[bpa(CH2)2O(CH2)2OH]Zn(NO3)](NO3) (1Zn), were comprehensively characterized and their X-ray single crystal structures have been determined. The complexes show hexacoordinated geometries, in which 1 acts as a tetradentate (1Cu) or pentadentate (1Co, 1Ni and 1Zn) ligand. DNA cleavage experiments have been performed on supercoiled double stranded DNA plasmids in order to compare the cleavage efficiency of all four metals in the same ligand environment of 1. In this assay, 1Co and 1Cu showed the highest cleavage efficiency, whereas 1Ni and 1Zn were virtually inactive. Quantification of the gel electrophoresis bands showed that more than 80% of the plasmid has suffered at least one single strand cut in the case of 1Cu, and about 50% of the plasmid was nicked by 1Co. The differential cleavage activity is discussed in relation to the structural findings and a mechanism is proposed for 1Cu.     

Co(II) and Ni(II) complexes with Schiff base ligands: Synthesis,characterization, and biological activity

The ligands, 1-acetylferrocenehydrazinecarboxamide (HL¹) and 1-acetylferrocenehydrazinecarbothioamide (HL²), and their Ni(II) and Co(II) complexes were synthesized. The properties of the synthesized compounds were determined by the elemental and spectroscopic analyses. Ni(II) and Co(II) acetates interact with the ligands at the molar ratios 1 : 1 and 1 : 2 to give coloured products. The complexes have octahedral geometry. The ligands are coordinated to Co(II) and Ni(II) centers via the azomethine nitrogen and thiolic sulfur /enolic oxygen atom. The ligands and their Co(II) and Ni(II) complexes were screened for antibacterial and antifungal activities. The Co(II) and Ni(II) complexes show enhanced inhibitory activity as compared to their parent ligands. The DNA cleavage activity of the Co(II) and Ni(II) complexes was determined by gel electrophoresis. It was shown that the complexes have better cleavage activity than the ligands. The antioxidant activity of the complexes was also evaluated and used to examine their scavenging ability on hydrogen peroxide.     

配合物生成反应速率常数与平衡常数之间的直线自由能关系IV:5位取代-1,10-邻菲 啉-铜(II)与镍(II)的金属交换反应动力学和机理研究

The kinetics of the metal exchange reactions between (5-R-phen)copper(II) (R = Me, H, Cl, and NO2) and Ni(II) was studied at 25?and ionic strength 1.0 mol dm-3 or pH 2.3-3.5. The rate of the exchange reactions was measured by a spectrophotometer. The reactions appeared to proceed through 3 different pathways which involved H+ attack and Ni attack as well as a pH- and Ni-independent dissociation of the complexes. The kinetics conforms to the following rate law: d[Ni(5-R-phen)]/dt = (kp + kH[H+] + kNi[Ni2+])[(Cu(5-R-phen)2+]. The reaction rate of the 3 pathways increased with decreasing basicity of the ligand. Some linear free energy relationships were found to exist between the reactivity of these Cu(II) complexes and the base strength of the ligand 5-R-phen. The mechanisms of the reactions are discussed.     

Artificial metalloprotease with active site comprising aldehyde group and Cu(II)cyclen complex

To design artificial proteases that cleave peptide backbones of a wide range of proteins at selected sites, artificial active sites comprising the Cu(II) complex of cyclen (Cu(II)Cyc) and aldehyde group were synthesized on a cross-linked polystyrene. The aldehyde group was employed as the binding site in view of its ability of reversible formation of imine bonds with epsilon-amino groups of Lys residues exposed on the surface of proteins and Cu(II)Cyc as the catalytic group for peptide hydrolysis. The two polymeric artificial metalloproteases synthesized in the present study cleaved all of the protein substrates examined (myoglobin, gamma-globulin, bovine serum albumin, human serum albumin, lysozyme, and ovalbumin), manifesting saturation kinetic behavior. At 50 degrees C and pH 9.0 or 9.5, K(m) was (1.3-22) x 10(-)(4) M, comparable to those of natural proteases, and k(cat) was (6.0-25) x 10(-)(4) s(-)(1), corresponding to half-lives of 4.6-19 min. Intermediacy of the imine complexes formed between the aldehyde group of the catalyst and the epsilon-amino groups of Lys residues of the substrates was confirmed by the trapping experiment with NaB(OAc)(3)H. MALDI-TOF MS of the proteolytic reaction mixtures revealed formation of various cleavage products. Structures of some of the cleavage products were determined by using carboxypeptidase A and trypsin. Among various cleavage sites thus identified, Gln(91)-Ser(92) and Ala(94)-Thr(95) were the major initial cleavage sites in the degradation of myoglobin by the two catalysts. The selective cleavage of Gln(91)-Ser(92) and Ala(94)-Thr(95) was attributed to general acid assistance in peptide cleavage by Tyr(146) located in proximity to the two peptide bonds. Broad substrate selectivity, high cleavage-site selectivity, and high proteolytic rate are achieved, therefore, by positioning the aldehyde group in proximity to Cu(II)Cyc attached to a cross-linked polystyrene.     

Tuning the activity of Zn(II) complexes in DNA cleavage: clues for design of new efficient metallo-hydrolases

The hydrolytic ability toward plasmid DNA of a mononuclear and a binuclear Zn(II) complex with two macrocyclic ligands, containing respectively a phenanthroline (L1) and a dipyridine moiety (L2), was analyzed at different pH values and compared with their activity in bis( p-nitrophenyl)phosphate (BNPP) cleavage. Only the most nucleophilic species [ZnL1(OH)]+ and [Zn2L2(OH)2]2+, present in solution at alkaline pH values, are active in BNPP cleavage, and the dinuclear L2 complex is remarkably more active than the mononuclear L1 one. Circular dichroism and unwinding experiments show that both complexes interact with DNA in a nonintercalative mode. Experiments with supercoiled plasmid DNA show that both complexes can cleave DNA at neutral pH, where the L1 and L2 complexes display a similar reactivity. Conversely, the pH-dependence of their cleavage ability is remarkably different. The reactivity of the mononuclear complex, in fact, decreases with pH while that of the dinuclear one is enhanced at alkaline pH values. The efficiency of the two complexes in DNA cleavage at different pH values was elucidated by means of a quantum mechanics/molecular mechanics (QM/MM) study on the adducts between DNA and the different complexed species present in solution.     

Red-light photosensitized cleavage of DNA by (l-lysine)(phenanthroline base)copper(II) complexes

Ternary copper(II) complexes [Cu(l-lys)B(ClO4)](ClO4)(1-4), where B is a heterocyclic base, viz. 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3) and dipyrido[3,2-a:2',3'-c]phenazene (dppz, 4), are prepared and their DNA binding and photo-induced DNA cleavage activity studied (l-lys =l-lysine). Complex 2, structurally characterized by X-ray crystallography, shows a square-pyramidal (4 + 1) coordination geometry in which the N,O-donor l-lysine and N,N-donor heterocyclic base bind at the basal plane and the perchlorate ligand is bonded at the elongated axial site. The crystal structure shows the presence of a pendant cationic amine moiety -(CH2)4NH3+ of l-lysine. The one-electron paramagnetic complexes display a d-d band in the range of 598-762 nm in DMF and exhibit cyclic voltammetric response due to Cu(II)/Cu(I) couple in the range of 0.07 to -0.20 V vs. SCE in DMF-Tris-HCl buffer. The complexes having phenanthroline bases display good binding propensity to the calf thymus DNA giving an order: 4 (dppz) > 3 (dpq) > 2 (phen)> 1 (bpy). Control cleavage experiments using pUC19 supercoiled DNA and distamycin suggest major groove binding for the dppz and minor groove binding for the other complexes. Complexes 2-4 show efficient DNA cleavage activity on UV (365 nm) or visible light (694 nm ruby laser) irradiation via a mechanistic pathway involving formation of singlet oxygen as the reactive species. The amino acid l-lysine bound to the metal shows photosensitizing effect at red light, while the heterocyclic bases are primarily DNA groove binders. The dpq and dppz ligands display red light-induced photosensitizing effects in copper-bound form.     

Toward efficient Zn(II)-based artificial nucleases

A series of cis-cis-triaminocyclohexane Zn(II) complex-anthraquinone intercalator conjugates, designed in such a way to allow their easy synthesis and modification, have been investigated as hydrolytic cleaving agents for plasmid DNA. The ligand structure comprises a triaminocyclohexane platform linked by means of alkyl spacers of different length (from C(4) to C(8)) to the anthraquinone group which may intercalate the DNA. At a concentration of 5 microM, the complex of the derivative with a C(8) alkyl spacer induces the hydrolytic stand scission of supercoiled DNA with a rate of 4.6 x 10(-6) s(-1) at pH 7 and 37 degrees C. The conjugation of the metal complex with the anthraquinone group leads to a 15-fold increase of the cleavage efficiency when compared with the anthraquinone lacking Zn-triaminocyclohexane complex. The straightforward synthetic procedure employed, allowing a systematic change of the spacer length, made possible to gain more insight on the role of the intercalating group in determining the reactivity of the systems. Comparison of the reactivity of the different complexes shows a remarkable increase of the DNA cleaving efficiency with the length of the spacer. In the case of too-short spacers, the advantages due to the increased DNA affinity are canceled due to the incorrect positioning of the reactive group, thus leading to cleavage inhibition.     

Design,synthesis, DNA binding ability,chemical nuclease activity and antimicrobial evaluation of Cu(II), Co(II), Ni(II) and Zn(II) metal complexes containing tridentate Schiff base

A novel Schiff base has been designed and synthesized using the bioactive ligand obtained from 4-aminoantipyrine, 3,4-dimethoxybenzaldehyde and 2-aminobenzoic acid. Its Cu(II), Co(II), Ni(II), Zn(II) complexes have also been synthesized in ethanol medium. The structural features have arrived from their elemental analyses, magnetic susceptibility, molar conductance, mass, IR, UV–Vis, ¹H NMR and ESR spectral studies. The data show that the complexes have composition of ML₂ type. The electronic absorption spectral data of the complexes suggest an octahedral geometry around the central metal ion. The interaction of the complexes with calf thymus (CT) DNA has been studied using absorption spectra, cyclic voltammetric, and viscosity measurement. The metal complexes have been found to promote cleavage of pUC19 DNA from the super coiled form I to the open circular form II. The complexes show enhanced antifungal and antibacterial activities compared with the free ligand.     

A simple copper(II)-L-histidine system for efficient hydrolytic cleavage of DNA

Copper(II)-L-histidine complexes effectively promote the cleavage of plasmid DNA and dideoxynucleotide dApdA at physiological pH and temperature. Studies of the mechanism of plasmid DNA cleavage by added radical scavengers, using rigorously anaerobic experiments, analyses for malondialdehyde-like products, religation assays, and HPLC analyses, indicate that DNA cleavage mediated by Cu(L-His) occurs via a hydrolytic path. The hydrolytic cleavage rate constants at 37 degrees C are estimated to be 0.76 h-1 for the decrease of form I and 0.25 h-1 for the increase of form III. The phosphoimager picture reveals that Cu(L-His) cleaves DNA with a certain sequence specificity (preferentially at 5'-GT-3'). The dinucleotide hydrolysis shows, with [Cu(L-His)] = 0.8 mM, rate enhancement factors of > 10(8). Interestingly, histidine-metal ion interactions (with Cu(II), Ni(II), Zn(II), etc.) have been used for various applications, e.g., protein purification, cross-linking, and targeting proteins to lipid bilayers. Our findings may provide the basis for developing new applications and new ways to design more effective and useful catalysts for DNA cleavage. Cu(L-His) is one of only a few well-defined metal complexes demonstrated to hydrolytically cleave dideoxynucleotides and DNA.     

[M(L)2(NCS)2] Complexes (M = Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II); L = Hydrazide of Diphenylphosphinylacetic Acid)

Dithiocyanates of bis(hydrazidediphenylphosphinylacetic acid) M(II), where M is Mn, Fe, Co, Ni, Cu, and Zn were synthesized. Their magnetic properties were studied. All compounds were paramagnetic monomers with pseudooctahedral stereochemistry, except for the Zn(II) complex.     

Studies of copper (Ⅱ) complexes as catalysts for the hydrolysis of DNA

Hydrolysis of DNA is an important enzymatic reaction , but it is exceedingly difficult to mimic in the laboratory because of the stability of hydrolysis of DNA. In this paper, the cleavage activity of complexes formed between Cu(Ⅱ) and four different amino acid or amino acid methyl ester on DNA is studied by gel elec-trophoresis. It is found that DNA could be cleaved by Cu(Ⅱ)-L-His and Cu(Ⅱ)-L-His methyl ester complexes and the efficiency of cleavage is largely dependent on the metal ion-to-ligand ratio. Further experiments show that the cleavage of DNA mediated by Cu(Ⅱ)-L-His complexes occurs via a hydrolytic mechanism and the active chemical species that affects DNA cleavage is proposed to be MI2H and ML2H22 .     

Co(II), Ni(II) and Cu(II) complexes with coumarin-8-yl Schiff-bases: spectroscopic, in vitro antimicrobial, DNA cleavage and fluorescence studies

A new series of Co(II), Ni(II) and Cu(II) complexes of the type ML·2H2O of Schiff-bases derived from m-substituted thiosemicarbazides and 8-acetyl-7-hydroxy-4-methylcoumarin have been synthesized and characterized by spectroscopic studies. Schiff-bases exhibit thiol-thione tautomerism wherein sulphur plays an important role in the coordination. The coordination possibility of the Schiff-bases towards metal ions have been proposed in the light of elemental analyses, spectral (IR, UV-vis, FAB-mass, ESR and fluorescence), magnetic and thermal studies. The low molar conductance values in DMF indicate that, the metal complexes are non-electrolytes. The cyclic voltammetric studies suggested that, the Cu(II) and Ni(II) complexes are of single electron transfer quasi-reversible nature. The Schiff-bases and its metal complexes have been evaluated for their in vitro antibacterial (Escherichia coli, Staphilococcus aureus, Bascillus subtilis and Salmonella typhi) and antifungal activities (Candida albicans, Cladosporium and Aspergillus niger) by MIC method. The Schiff-base I and its metal complexes exhibited DNA cleavage activity on isolated DNA of A. niger.     

Influence of several factors on potential-modulated DNA cleavage by the Cu(en)2 and Cu(EDTA) complexes

Potential-modulated DNA cleavage in the presence of copper–ethylenediamine (en) and –ethylenediamine tetraacetic acid (EDTA) complexes was investigated at a gold electrode in a thin layer cell. DNA can be efficiently cleaved through production of active oxygen species at −0.50 V (vs. Ag/AgCl/KCl(sat)) by reducing Cu(en)₂²⁺ to Cu(en)₂⁺ or Cu(EDTA)²⁻ to Cu(EDTA)³⁻. The extent of DNA cleavage increased as the working potential was shifted more negative and the electrolysis time was increased in air-saturated solution. When a small flow of O₂ was passed through the solution during electrolysis, the extent of DNA cleavage was dramatically enhanced. In the absence of Cu(en)₂²⁺ or Cu(EDTA)²⁻ complex, slight DNA cleavage was observed at a more negative working potential due to the reduction of oxygen at the electrode. This observation suggests that potential-modulated DNA cleavage was caused mainly by electrochemical reduction of the Cu(en)₂²⁺ or Cu(EDTA)²⁻ complex in the presence of oxygen. The cleaved DNA fragments were separated by high performance liquid chromatography (HPLC). The experimental results proved that this method of potential-modulated DNA cleavage by Cu(en)₂²⁺ and Cu(EDTA)²⁻ complexes is simple, mild and highly efficient.     

Efficient plasmid DNA cleavage by a mononuclear copper(II) complex

The Cu(II) complex of the ligand all-cis-2,4,6-triamino-1,3,5-trihydroxycyclohexane (TACI) is a very efficient catalyst of the cleavage of plasmid DNA in the absence of any added cofactor. The maximum rate of degradation of the supercoiled plasmid DNA form, obtained at pH 8.1 and 37 degrees C, in the presence of 48 microM TACI.Cu(II), is 2.3 x 10(-3) s(-1), corresponding to a half-life time of only 5 min for the cleavage of form I (supercoiled) to form II (relaxed circular). The dependence of the rate of plasmid DNA cleavage from the TACI.Cu(II) complex concentration follows an unusual and very narrow bell-like profile, which suggests an high DNA affinity of the complexes but also a great tendency to form unreactive dimers. The reactivity of the TACI.Cu(II) complexes is not affected by the presence of several scavengers for reactive oxygen species or when measured under anaerobic conditions. Moreover, no degradation of the radical reporter Rhodamine B is observed in the presence of such complexes. These results are consistent with the operation of a prevailing hydrolytic pathway under the normal conditions used, although the failure to obtain enzymatic religation of the linearized DNA does not allow one to rule out the occurrence of a nonhydrolytic oxygen-independent cleavage. A concurrent oxidative mechanism becomes competitive upon addition of reductants or in the presence of high levels of molecular oxygen: under such conditions, in fact, a remarkable increase in the rate of DNA cleavage is observed.     

Spectral and thermal studies of 4-(1H-pyrazolo (3,4-d) pyrimidin-4-ylazo) benzene-1,3-diol complexes of cobalt(II), nickel(II) and copper(II)

4-(1H-Pyrazolo (3,4-d) pyrimidin-4-ylazo) benzene-1,3-diol was synthesized and characterized by various spectral and analytical techniques. Semiempirical quantum calculations using the AM1 method have been performed in order to evaluate the geometry and electronic structure of the title azodye in the ground state. The complex formation between Co(II), Ni(II) and Cu(II) ions and the title azodye was studied conductometrically and spectrophotometrically. The spectrophotometric determination of the title metal ions and titration using EDTA are reported. Co(II), Ni(II) and Cu(II) complexes of the title azodye have been synthesized and characterized by elemental analysis, conductivity, magnetic susceptibility, IR, UV-Vis and thermal analysis (TGA and DTA).The spectral and magnetic data suggested the octahedral geometry for Co(II) and Ni(II) complexes while Cu(II) complexes have square planar geometry. The thermal studies confirmed the chemical formulations of the title complexes. The thermal degradation takes place in two or three steps depending on the type of the metal and the geometry of the complexes. The kinetics of the decomposition was examined by using Coats-Redfern relation. The activation energies and other activation parameters (DeltaH, DeltaS and DeltaG) were computed and related to the bonding and stereochemistry of the complexes.     

The ATCUN domain as a probe of intermolecular interactions: application to calmodulin-peptide complexes

The utility of a three-residue Cu2+ binding motif (ATCUN domain) for studying intermolecular interactions is demonstrated. By comparing a set of 1H-15N correlation spectra recorded on complexes of calmodulin (CaM) and peptides with the ATCUN tag in the presence and absence of Cu2+ the two possible canonical binding orientations of the peptide can be rapidly distinguished. The methodology is confirmed with studies of complexes of CaM and peptides from myosin light chain kinase and CaM kinase kinase, for which high-resolution structures are available, and then applied to a complex with CaM kinase I for which structural data has not been obtained. The orientation of the CaM kinase I and myosin light chain kinase peptides are shown to be identical. In the case of a complex of CaM with a peptide for which structural information is not available, the present methodology, in combination with 1H-15N residual dipolar couplings measured on CaM, and the database of existing CaM-peptide structures, allows a homology model to be built rapidly and with confidence.