TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)     

Bcl-rambo induces apoptosis via interaction with the adenine nucleotide translocator

The Bcl-2 family proteins plays a central role in apoptosis. The pro- or anti-apoptotic activities of Bcl-2 family are dependent on the Bcl-2 homology (BH) regions. Bcl-rambo, a new pro-apoptotic member, is unusual in that its pro-apoptotic activity is independent of its BH domains. However, the mechanism underlying Bcl-rambo-induced apoptosis is largely unknown. Mitochondrial localization is indispensable for the pro-apoptotic function of Bcl-rambo. Bcl-rambo interacts physically with the adenine nucleotide translocator (ANT), suppresses the ADT/ATP-dependent translocation activity of ANT. Collectively, our data indicate Bcl-rambo is a pro-apoptotic member of the Bcl-2 family, induces the permeability transition via interaction with ANT.

Structured summary of protein interactions:

Bcl-Rambo and HSP60colocalize by fluorescence microscopy (View interaction)Bcl-rambobinds to ANT1 by pull down (View interaction)     

The C-terminal domain of human Rev1 contains independent binding sites for DNA polymerase η and Rev7 subunit of polymerase ζ

Human Rev1 is a translesion synthesis (TLS) DNA polymerase involved in bypass replication across sites of DNA damage and postreplicational gap-filling. Rev1 plays an essential structural role in TLS by providing a binding platform for other TLS polymerases that insert nucleotides across DNA lesions (polη, polι, polκ) and extend the distorted primer-terminus (pol?). We use NMR spectroscopy to demonstrate that the Rev1 C-terminal domain utilizes independent interaction interfaces to simultaneously bind a fragment of the ’inserter’ polη and Rev7 subunit of the ’extender’ pol?, thereby serving as a cassette that may accommodate several polymerases making them instantaneously available for TLS.

Structured summary of protein interactions

REV1, REV3 and REV7physically interact by nuclear magnetic resonance (View interaction), molecular sieving (View interaction) and isothermal titration calorimetry (View interaction).REV3 and REV7bind by molecular sieving (View interaction)REV1 and Polη-RIR peptidebind by nuclear magnetic resonance (View interaction)REV1, REV3, REV7 and Polη-RIR peptidephysically interact by nuclear magnetic resonance (View interaction).     

Characterizing the neurite outgrowth inhibitory effect of Mani

Mani (myelin-associated neurite-outgrowth inhibitor) protein is implicated in both axonal guidance and axonal regeneration after central nervous system (CNS) injury. Here, we applied a neurite outgrowth assay, coupled with a siRNA-driven investigation and immunocytochemistry, to unveil Mani’s axonal outgrowth inhibitory effect in embryonic rat cortical primary neurons in vitro. We further demonstrate Mani’s neuronal localization in comparison with a principal subunit, Cdc27, of the anaphase promoting complex (APC). Considering the protein structure of Mani obtained via a series of bio-computational studies, we propose a Cdc27-Mani-APC-related signalling pathway may be involved in CNS axon regeneration.

Structured summary of protein interactions

MANIphysically interacts with DAZAP2 by two hybrid (View interaction)MANIphysically interacts with FAM168A by two hybrid (View interaction)MANIphysically interacts with YPEL2 by two hybrid (View interaction)MANIphysically interacts with TMTC1 by two hybrid (View interaction)     

Genome-wide biochemical analysis of Arabidopsis protein phosphatase using a wheat cell-free system

Plant genome possesses over 100 protein phosphatase (PPase) genes that are key regulators of signal transduction via phosphorylation/dephosphorylation event. Here we report a comprehensive functional analysis of protein serine/threonine, dual-specificity and tyrosine phosphatases using recombinant PPases produced by wheat cell-free protein synthesis system. Eighty-two recombinant PPases were successfully produced using Arabidopsis full-length cDNA as templates. In vitro PPase assay was performed using phosphorylated myelin basic protein as substrate. Among the AtPPases examined, 26 serine/threonine, three dual-specificity and one tyrosine PPases exhibited catalytic activity, including 20 serine/threonine and one dual-specificity PPases that showed in vitro activities for the first time. Our study demonstrates genome-wide biochemical analysis of AtPPases using wheat cell-free system, and provides new information and insights on enzyme activities.

Structured summary of protein interactions

PTP1dephosphorylatesMBP by phosphatase assay (View interaction).AtPP2CdephosphorylatesMBP by phosphatase assay (View interaction).POLTEdephosphorylatesMBP by phosphatase assay (View interaction).TOPP8dephosphorylatesMBP by phosphatase assay (View interaction).HAB1dephosphorylatesMBP by phosphatase assay (View interaction).ABI2dephosphorylatesMBP by phosphatase assay (View interaction).At1g34750dephosphorylatesMBP by phosphatase assay (View interaction).At1g43900dephosphorylatesMBP by phosphatase assay (View interaction).At3g15260dephosphorylatesMBP by phosphatase assay (View interaction).At5g53140dephosphorylatesMBP by phosphatase assay (View interaction).At1g18030dephosphorylatesMBP by phosphatase assay (View interaction).At3g06270dephosphorylatesMBP by phosphatase assay (View interaction).At2g25070dephosphorylatesMBP by phosphatase assay (View interaction).At3g02750dephosphorylatesMBP by phosphatase assay (View interaction).At5g10740dephosphorylatesMBP by phosphatase assay (View interaction).at4g26080dephosphorylatesMBP by phosphatase assay (View interaction).At4g28400dephosphorylatesMBP by phosphatase assay (View interaction).At5g06750dephosphorylatesMBP by phosphatase assay (View interaction).At4g31860dephosphorylatesMBP by phosphatase assay (View interaction).At3g17250dephosphorylatesMBP by phosphatase assay (View interaction).At4g38520dephosphorylatesMBP by phosphatase assay (View interaction).At3g05640dephosphorylatesMBP by phosphatase assay (View interaction).At5g66080dephosphorylatesMBP by phosphatase assay (View interaction).At1g79630dephosphorylatesMBP by phosphatase assay (View interaction).At2g30170dephosphorylatesMBP by phosphatase assay (View interaction).At5g24940dephosphorylatesMBP by phosphatase assay (View interaction).     

The crystal structure of galactitol-1-phosphate 5-dehydrogenase from Escherichia coli K12 provides insights into its anomalous behavior on IMAC processes

Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni²⁺-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn²⁺- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag.

Structured summary of protein interactions

GPDH and GPDHbind by molecular sieving (View interaction)GPDH and GPDHbind by x-ray crystallography (View interaction)GPDH and GPDHbind by cosedimentation in solution (View interaction)     

Characterization of a Wnt-binding site of the WIF-domain of Wnt inhibitory factor-1

A Wnt-binding site of the WIF-domain of Wnt inhibitory factor-1 was localized by structure-guided arginine-scanning mutagenesis in combination with surface plasmon resonance assays. Our observation that substitution of some residues of WIF resulted in an increased affinity for Wnt5a, but decreased affinity for Wnt3a, suggests that these residues may define the specificity spectrum of WIF for Wnts. These results hold promise for a more specific targeting of Wnt family members with WIF variants in various forms of cancer.

Structured summary of protein interactions

WIFbinds to Wnt7a by surface plasmon resonance (View Interaction)WIFbinds to Wnt4 by surface plasmon resonance (View Interaction)WIF and Wnt3aphysically interact by competition binding (View Interaction 1, 2, 3, 4,5, 6)WIFbinds to Wnt9b by surface plasmon resonance (View Interaction)WIFbinds to Wnt5a by surface plasmon resonance (View Interaction)WIFbinds to Wnt11 by surface plasmon resonance (View Interaction)WIFbinds to Wnt3a by surface plasmon resonance (View Interaction)Wnt-5a and WIFphysically interact by competition binding (View Interaction 1,2, 3, 4, 5, 6)     

Arabidopsis Zinc Ribbon 3 is the ortholog of yeast mitochondrial HSP70 escort protein HEP1 and belongs to an ancient protein family in mitochondria and plastids

ZR proteins belong to a phylogenetically conserved family of small zinc-ribbon proteins in plastids and mitochondria of higher plants. The function of these proteins is so far unclear. The mitochondrial proteins share sequence similarities with mitochondrial Hsp70 escort proteins (HEP) from Saccharomyces cerevisiae (HEP1) and human. Expression of the mitochondrial ZR protein from Arabidopsis, ZR3, rescued a hep1 knockout mutant from yeast. Accordingly, ZR3 was found to physically interact with mitochondrial Hsp70 from Arabidopsis. Our findings support the idea that mitochondrial and plastidic ZR proteins from higher plants are orthologs of HEP proteins.

Structured summary of protein interactions

ZR3physically interacts with mtHSC70-2 by pull down (View interaction)ZR3physically interacts with mtHSC70-1 by pull down (View interaction)     

Synthesis of organophosphorus compounds containing different Y,C,Y-chelating ligands. Crystal structure of P ← N intramolecularly coordinated diselenoxophosphorane

The reactions of L^1-3Li salts containing different Y,C,Y-chelating ligands L¹ = 2,6-(t-BuOCH₂)₂C₆, L² = 2,6-(MesOCH₂)₂C₆ and L³ = 2,6-(Me₂NCH₂)₂C₆ with PCl₃ is reported. While the presence of ligands L^2,3 afforded the synthesis of dichlorophosphines L²PCl₂ (2) and L³PCl₂ (3), the use of ligand L¹ resulted to the isolation of O → P coordinated 1-chloro-7-(t-butoxymethyl)-3H-2,1-benzoxaphosphole (1) as the result of the cyclization type reaction of dichlorophosphines L¹PCl₂. The hydrolysis of compounds 1-3 as well as the preparation of phosphanes L²PH₂ (7), L³PH₂ (8), L²PH(SnMe₃) (9) and L³PH(SnMe₃) (10) is also discussed. The presence of N → P coordination enabled the isolation of N → P coordinated diselenoxophosphorane L³PSe₂ (11). Compounds 1-11 were characterized by the help of multinuclear NMR spectroscopy, ESI mass spectrometry and the structure of compound 11 was established by X-ray diffraction analysis.     

Molecular structures of nickel(II) monochelates of a racemic tridentate ligand and co-ligand induced structural variations

Using a racemic mixture of the tridentate ligand, (((2-pyridyl)ethylamine)methyl)phenolate ion (L⁻) and , NCS⁻, (NC)₂N⁻, OAc⁻ as coligands, complexes having the formula [Ni(L)(N₃)] (1), [Ni(L)(NCS)]₂ (2), [Ni₂(L)₂(OAc)(N(CN)₂)]_n (3) were prepared and structurally characterized. In 1, Ni(II) has a square planar geometry and phenolate oxygen is involved in dipolar ?N^δ+ interaction with electrophilic central nitrogen atom of coordinated azide ion. Complex 2 is dimeric in nature and nickel(II) is penta-coordinated. Compounds 1 and 2 exist as centrosymmetric dimers made up of a pair of R and S enantiomers of L. In 3, an acetate and phenoxo bridged dinickel complex is present which is further linked to a zig-zag coordination polymer by the dicyanamide ion. In a given chain of 3, both L have same enantiomeric form and either RR or SS dimers are repeated along the chain. The magnetic properties are described.     

Mn(II) coordination architectures with mixed ligands of 3-(2-pyridyl)pyrazole and carboxylic acids bearing different secondary coordination donors and pendant skeletons

In our efforts to investigate the factors that affect the formation of coordination architectures, such as secondary coordination donors and pendant skeletons of the carboxylic acid ligands, as well as H-bonding and other weak interactions, two kinds of ligands: (a) 3-(2-pyridyl)pyrazole (L¹) with a non-coordinated N atom as a H-bonding donor, a 2,2′-bipyridyl-like chelating ligand, and (b) four carboxylic ligands with different secondary coordination donors and/or pendant skeletons, 1,4-benzenedicarboxylic acid (H₂L²), 4-sulfobenzoic acid (H₂L³), quinoline-4-carboxylic acid (HL⁴) and fumaric acid (H₂L⁵), have been selected to react with Mn(II) salts, and five new complexes, [Mn(L¹)₂(SO₄)]₂ (1), [Mn(L¹)₂(L²)]_∞ (2), [Mn(L¹)(HL³)₂]_∞ (3), Mn(L¹)₂(L⁴)₂ (4), and [Mn(L¹)₂(L⁵)]_∞ (5), have been obtained and structurally characterized. The structural differences of 1-5 can be attributed to the introduction of the different carboxylic acid ligands (H₂L², H₂L³, HL⁴, and H₂L⁵) with different secondary coordination donors and pendant skeletons, respectively. This result also reveals that the typical H-bonding (i.e. N-H?O and O-H?O) and some other intra- or inter-molecular weak interactions, such as C-H?O weak H-bonding and π?π interactions, often play important roles in the formation of supramolecular aggregates, especially in the aspect of linking the multi-nuclear discrete subunits or low-dimensional entities into high-dimensional supramolecular networks.     

Subcellular localization of adenylate kinases in Plasmodium falciparum

Adenylate kinases (AK) play a key role in nucleotide signaling processes and energy metabolism by catalyzing the reversible conversion of ATP and AMP to 2 ADP. In the malaria parasite Plasmodium falciparum this reaction is mediated by AK1, AK2, and a GTP:AMP phosphotransferase (GAK). Here, we describe two additional adenylate kinase-like proteins: PfAKLP1, which is homologous to human AK6, and PfAKLP2. Using GFP-fusion proteins and life cell imaging, we demonstrate a cytosolic localization for PfAK1, PfAKLP1, and PfAKLP2, whereas PfGAK is located in the mitochondrion. PfAK2 is located at the parasitophorous vacuole membrane, and this localization is driven by N-myristoylation.

Structured summary of protein interactions

EXP-1 and PfAK2colocalize by fluorescence microscopy (View interaction)PfAK2 and SERPcolocalize by fluorescence microscopy (View interaction)     

STAT3 repressed USP7 expression is crucial for colon cancer development

Interleukin-6 (IL-6) induced STAT3 activation is viewed as crucial for multiple tumor growth and metastasis, including colon cancer. However, the molecular mechanisms remain largely unexplored. Here, we show that expression of ubiquitin-specific protease 7 (USP7), a deubiquitylating enzyme, is decreased in STAT3-positive tumors. IL-6 administration or transfection of a constitutively activated STAT3 in SW480 cells also repressed USP mRNA expression. Using luciferase reporter and ChIP assay, we found that STAT3 bound to the promoter region of USP7 and inhibited its activity through recruiting HDAC1. As a result of the decline of USP7 expression, endogenous P53 protein level was decreased. Thus, our results suggest a previously unknown STAT3-USP7-P53 molecular network controlling colon cancer development.

Structured summary of protein interactions:

STAT3physically interacts with HDAC1 by anti bait coimmunoprecipitation (View interaction)     

Study of the coordination behaviour of (3,5-diphenyl-1H-pyrazol-1-yl)ethanol against Pd(II), Zn(II) and Cu(II)

A new easily synthetic route with a 96% yield of ligand 2-(3,5-diphenyl-1H-pyrazol-1-yl)ethanol (L) is obtained. The reactivity of L against Pd(II), Zn(II) and Cu(II) leads to [PdCl₂(L)₂] (1), [ZnCl₂(L)] (2) and [CuCl(L′)]₂ (3) (L′ is the ligand L without alcoholic proton), respectively. According to the different geometries imposed by the metallic centre and the capability of L to present various coordination links, it has been obtained complexes with square planar (1 and 3) or tetrahedral (2) geometry and different nuclearity: monomeric (1 and 2) or dimeric (3). Complete characterisation by analytical and spectroscopic methods, resolution of L and 1-3 by single-crystal X-ray diffraction and magnetic studies for complex 3 are presented.     

(Pyrazolylmethyl)pyridine platinum(II) and gold(III) complexes: Synthesis, structures and evaluation as anticancer agents

Reactions of 2-(3,5-dimethylpyrazol-1-ylmethyl)pyridine (L1), 2-(3,5-diphenylpyrazol-1-ylmethyl)pyridine (L2), 2-(3,5-di-tert-butylpyrazol-1-ylmethyl)pyridine (L3) and 2-(3-p-tolylpyrazol-1-ylmethyl)pyridine (L4) with K₂[PtCl₄] in a mixture of ethanol and water formed the dichloro platinum complexes [PtCl₂(L1)] (1), [PtCl₂(L2)] (2), [PtCl₂(L3)] (3) and [PtCl₂(L4)] (4). Complex 1, [PtCl₂(L1)], could also be prepared in a mixture of acetone and water. Performing the reactions of L2 and L3 in a mixture of acetone and water, however, led to C-H activation of acetone under mild conditions to form the neutral acetonyl complexes [Pt(CH₂COCH₃)Cl(L2)] (2a) and [Pt(CH₂COCH₃)Cl(L3)] (3a). The same ligands reacted with HAuCl₄ · 4H₂O in a mixture of ethanol and water to form the gold salts [AuCl₂(L1)][AuCl₄] (5) [AuCl₂(L2)][Cl] (6) [AuCl₂(L3)][Cl] (7) and [AuCl₂(L4)][AuCl₄] (8); however, with the pyrazolyl unit in the para position of the pyridinyl ring in 4-(3,5-dimethylpyrazol-1-ylmethyl)pyridine (L5), 4-(3,5-diphenylpyrazol-1-ylmethyl)pyridine (L6) neutral gold complexes [AuCl₃(L5)] (9) and [AuCl₂(L6)] (10) were formed; signifying the role the position of the pyrazolyl group plays in product formation in the gold reactions. X-ray crystallographic structural determination of L6, 2, 33a, 8 and 10 were very important in confirming the structures of these compounds; particularly for 3a and 8 where the presence of the acetonyl group confirmed C-H activation and for 8 where the counter ion is . Cytotoxicity studies of L2, L4 and complexes 1-10 against HeLa cells showed the Au complexes were much less active than the Pt complexes.     

Complexation of iron(III) by catecholate-type polyphenols

We report here a complete physico-chemical study of the chelation of iron(III) by catechin (L¹), an abundant polyphenol in green tea. Using a fruitful combination of electrospray mass spectrometry, absorption spectrophotometry and potentiometry, we have characterized three ferric complexes of catechin (L¹Fe, and (L¹)₃Fe) as well as a ternary complex L¹FeNTA when an exogenous ligand (nitrilotriacetic acid) is added to the medium. Thanks to this study, we discuss the influence of an exogenous tetradentate ligand in the ferric recognition processes by catecholate-type polyphenols.     

Pd(II) complexes containing N-alkyl-3-pyridine-5-trifluoromethyl pyrazole ligands: Synthesis, NMR studies and X-ray crystal structures

Palladium [PdCl₂(L)] complexes with N-alkylpyridylpyrazole derived ligands [2-(5-trifluoromethyl-1H-pyrazol-3-yl)pyridine (L¹), 2-(1-ethyl-5-trifluoromethyl-1H-pyrazol-3-yl)pyridine (L²), 2-(1-octyl-5-trifluoromethyl-1H-pyrazol-3-yl)pyridine (L³), and 2-(3-pyridin-2-yl-5-trifluoromethyl-pyrazol-1-yl)ethanol (L⁴) were synthesised. The crystal and molecular structures of [PdCl₂(L)] (L = L², L³, L⁴) were resolved by X-ray diffraction, and consist of monomeric cis-[PdCl₂(L)] molecules. The palladium centre has a typical square-planar geometry, with a slight tetrahedral distortion. The tetra-coordinate metal atom is bonded to one pyridinic nitrogen, one pyrazolic nitrogen and two chlorine ligands in cis disposition. Reaction of L (L², L⁴) with [Pd(CH₃CN)₄](BF₄)₂, in the ratio 1M:2L, gave complexes [Pd(L)]₂(BF₄)₂. Treatment of [PdCl₂(L)] (L = L², L⁴) with NaBF₄ and pyridine (py) and treatment of the same complexes with AgBF₄ and triphenylphosphine (PPh₃) yielded [Pd(L)(py)₂](BF₄)₂ and [Pd(L)(PPh₃)₂](BF₄)₂ complexes, respectively. Finally, reaction of [PdCl₂(L⁴)] with 1 equiv of AgBF₄ yields [PdCl(L⁴)](BF₄).     

Zinc complexes of a new N3O ligand and their biomimetic reactions

The new N₃O ligand 2-(pyridylmethylamino)-3-(3,5-dimethylpyrazol-1-yl)-propionic acid (L1H) was synthesized and converted to L1Zn-Cl and L1Zn-Br. These complexes are tetrameric in the solid state with bridging carboxylate functions. The reaction of deprotonated L1H with zinc nitrate or zinc perchlorate yielded the aqua complexes [L1Zn-OH₂] X with and , which crystallize as carboxylate-bridged dimers. Their deprotonation produced, in situ, the hydroxide complex L1Zn-OH, which acted as a base toward p-nitrophenol and bis(p-nitrophenyl)phosphoric acid resulting in L1Zn-ONit and L1Zn-OPO(ONit)₂. Tris(p-nitrophenyl)phosphate was cleaved hydrolytically by L1Zn-OH, releasing one p-nitrophenyl group. A kinetic investigation of this cleavage reaction under pseudo-first-order conditions has yielded second-order rate constants k″ of 0.9 s⁻¹ M⁻¹ in 50% aqueous DMSO and 4.0 s⁻¹ M⁻¹ in 75% aqueous DMSO.     

New ternary copper(II) complexes of l-alanine and heterocyclic bases: DNA binding and oxidative DNA cleavage activity

Four new ternary copper(II) complexes of α-amino acid having polypyridyl bases of general formulation [Cu(l-ala)(B)(H₂O)](X) (1-4), where l-ala is l-alanine, B is an N,N-donor heterocyclic base, viz. 2,2′-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2) and 5,6-phenanthroline dione (dione, 3), dipyrido[3,2:2′,3′-f]quinoxaline (dpq, 4), and X = / are synthesized, characterized by various spectroscopic and X-ray crystallographic methods. The complexes show a distorted square-pyramidal (4 + 1) CuN₃O₂ coordination geometry. The one-electron paramagnetic complexes (1-4) display a low energy d-d band near 600 nm in aqueous medium and show a quasi-reversible cyclic voltammetric response due to one-electron Cu(II)/Cu(I) reduction near −100 mV (versus SCE) in DMF-0.1 M TBAP. Binding interactions of the complexes with calf thymus DNA (CT-DNA) were investigated by UV-Vis absorption titration, ethidium bromide displacement assay, viscometric titration experiment and DNA melting studies. All the complexes barring the complexes 1 and 3 are avid binder to the CT-DNA in the DNA minor groove giving an order: 4 > 2 ? 1, 3. The complexes 2 and 4 show appreciable chemical nuclease activity in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent. Hydroxyl radical was investigated to be the DNA cleavage active species. Control experiments in the presence of distamycin-A show primarily minor groove-binding propensity for the complexes 2 and 4 to the DNA.