NMR structure of AbhN and comparison with AbrBN: FIRST insights into the DNA binding promiscuity and specificity of AbrB-like transition state regulator proteins

Understanding the molecular mechanisms of transition state regulator proteins is critical, since they play a pivotal role in the ability of bacteria to cope with changing environments. Although much effort has focused on their genetic characterization, little is known about their structural and functional conservation. Here we present the high resolution NMR solution structure of the N-terminal domain of the Bacillus subtilis transition state regulator Abh (AbhN), only the second such structure to date. We then compare AbhN to the N-terminal DNA-binding domain of B. subtilis AbrB (AbrBN). This is the first such comparison between two AbrB-like transition state regulators. AbhN and AbrBN are very similar, suggesting a common structural basis for their DNA binding. However, we also note subtle variances between the AbhN and AbrBN structures, which may play important roles in DNA target specificity. The results of accompanying in vitro DNA-binding studies serve to highlight binding differences between the two proteins.     

Novel DNA binding domain and genetic regulation model of Bacillus subtilis transition state regulator abrB

We have determined the high resolution NMR solution structure of the novel DNA binding domain of the Bacillus subtilis transition state regulator AbrB. Comparisons of the AbrB DNA binding domain with DNA binding proteins of known structure show that it is a member of a completely novel class of DNA recognition folds that employs a dimeric topology for cellular function. This new DNA binding conformation is referred to as the looped-hinge helix fold. Sequence homology investigations show that this DNA binding topology is found in other disparately related microbes. Structural analysis of the AbrB DNA binding domain together with bioanalytical and mutagenic data of full length AbrB allows us to construct a general model that describes the genetic regulation properties of AbrB.     

Interaction of two new mixed ligand copper(II) complexes with DNA probed by thermodynamic and spectroscopic studies

The DNA binding behavior of [Cu(4,7-dmp)(phen-dione)Cl]Cl (1) and [Cu(2,9-dmp)(phen-dione)Cl]Cl (2) where dmp and phen-dion stand for dimethyl-1,10-phenanthroline and 1,10-phenanthroline-5,6-dion, respectively, was studied with a series of techniques including Viscometry, UV–Vis absorption, circular dichroism and fluorescence spectroscopy. Cytotoxicity effect was also investigated. Thermodynamic parameters, enthalpy and entropy changes were calculated according to Van’t Hoff equation, which indicated that both reactions are predominantly enthalpically driven. However, these two complexes show different behavior in fluorescence, circular dichroism and viscometry methods which indicate the Cu(II) complexes interact with calf-thymus DNA by different mode of binding. These have further been verified by competition studies using Hoechst as a distinct groove binder. All these results indicate that these two complexes (1) and (2) interact with CT-DNA via groove binding and partially intercalative mode, respectively and the binding affinity of the complex 1 is higher than that of complex 2. Finally, our findings suggest that the type of ligands and structure of complexes have marked effect on the binding affinity of complexes involving CT-DNA. Also, these new complexes showed excellent antitumor activity against human T lymphocyte carcinoma-Jurkat cell line.     

Structural studies on ligand–DNA systems: A robust approach in drug design

Molecular docking, molecular mechanics, molecular dynamics and relaxation matrix simulation protocols have been extensively used to generate the structural details of ligand-receptor complexes in order to understand the binding interactions between the two entities. Experimental methods like NMR spectroscopy and X-ray crystallography are known to provide structural information about ligand-receptor complexes. In addition, fluorescence spectroscopy, circular dichroism (CD) spectroscopy and molecular docking have also been utilized to decode the phenomenon of the ligand-DNA interactions, with good correlation between experimental and computational results. The DNA binding affinity was demonstrated by analysing fluorescence spectral data. Structural rigidity of DNA upon ligand binding was identified by CD spectroscopy. Docking is carried out using the DNA-Dock program which results in the binding affinity data along with structural information like interatomic distances and H-bonding, etc. The complete structural analyses of various drug-DNA complexes have afforded results that indicate a specific DNA binding pattern of these ligands. It also exhibited that certain structural features of ligands can make a ligand to be AT- or GC-specific. It was also demonstrated that changing specificity from AT base pairs to GC base pairs further improved the DNA topoisomerase inhibiting activity in certain ligands. Thus, a specific molecular recognition signature encrypted in the structure of ligand can be decoded and can be effectively employed in designing more potent antiviral and antitumour agents.     

Spectroscopic studies on the interaction of quercetin-terbium(III) complex with calf thymus DNA

The interaction of native calf thymus DNA (CT-DNA) with quercetin-terbium(III) [Q-Tb(III)] complex at physiological pH was monitored by UV absorption spectrophotometry, circular dichroism, fluorescence spectroscopy, and viscosimetric techniques. The complex displays binding properties to the CT-DNA and was found to interact with CT-DNA through outside binding, demonstrated by a hypochromic effect of Q-Tb(III) on the UV spectra of CT-DNA and the calculated association constants (K). Also, decrease in the specific viscosity of CT-DNA, decrease in the fluorescence intensity of Q-Tb(III) solutions in the presence of increasing amounts of CT-DNA, and detectable changes in the circular dichroism spectrum of CT-DNA are other evidences to indicate that Q-Tb(III) complex interact with CT-DNA through outside binding.     

Macromolecular assembly of the transition state regulator AbrB in its unbound and complexed states probed by microelectrospray ionization mass spectrometry

The Bacillus subtilis global transition-state regulator AbrB specifically recognizes over 60 different DNA regulatory regions of genes expressed during cellular response to suboptimal environments. Most interestingly the DNA regions recognized by AbrB share no obvious consensus base sequence. To more clearly understand the functional aspects of AbrB activity, microelectrospray ionization mass spectrometry has been employed to resolve the macromolecular assembly of unbound and DNA-bound AbrB. Analysis of the N-terminal DNA binding domain of AbrB (AbrBN53, residues 1-53) demonstrates that AbrBN53 is a stable dimer, showing no apparent exchange with a monomeric form as a function of pH, ionic strength, solvent, or protein concentration. AbrBN53 demonstrates a capacity for DNA binding, underscoring the role of the N-terminal domain in both DNA recognition and dimerization. Full-length AbrB is shown to exist as a homotetramer. An investigation of the binding of AbrBN53 and AbrB to the natural DNA target element sinIR shows that AbrBN53 binds as a dimer and AbrB binds as a tetramer. This study represents the first detailed characterization of the stoichiometry of a transition-state regulator binding to one of its target promoters.     

Molecular characterization of the transition state regulator AbrB from Bacillus stearothermophilus

The Bacillus subtilis transition state regulator AbrB(su) is a DNA-binding protein that acts on several genes either as activator, repressor, or preventer. However, among genes under its control, neither common binding sites could be identified nor could the structural features of this broad and specific interaction be elucidated. Attempts to elucidate these interesting features by crystallizing AbrB(su) have failed so far. Therefore, to solve this problem, we focused in this work on identifying an AbrB(su) homologue from Bacillus stearothermophilus. Using a novel method, the entire abrB(st) gene of B. stearothermophilus was cloned and sequenced. The gene encodes a 95 amino acid protein that shows 77% identity and 85% similarity to the mesophilic B. subtilis protein. A calmodulin binding peptide-tagged fusion of the thermophilic gene was constructed for overexpression and efficient affinity column purification of the AbrB(st) protein. The purified protein showed, after removal of the tag, an oligomerization behavior through hexamer formation that is essential for its DNA binding activity.     

Interaction of ethidium bromide with DNA. Optical and electrooptical study

The binding of ethidium bromide to DNA has been studied by various optical methods. From fluorescence polarization studies, and film, electric linear dichroism, and circular dichroism spectra, we propose assignments of the absorption bands of the dye, which are discussed in connection with wave-mechanical calculations recently reported. The optical activity induced in the dye absorption bands upon binding to DNA was attributed to various origins depending on the electronic transition considered. The visible absorption band displayed a circular dichroism due to the asymmetry of the binding site and independent of the amount of binding. The transition identified at 378 nm from the circular dichroism and electric dichroism observations was thought to be due to a magnetic-dipole transition. It remained constant with increasing amounts of dye bound. The main ultraviolet band showed circular dichroism characteristics corresponding to exciton interactions between dye molecules bound to neighboring sites. The electric dichroism observed for the strongly bound dye molecules indicated that the phenanthridinium ring of ethidium bromide was probably not perfectly parallel to the DNA base planes. When the amount of dye bound to DNA exceeded the maximum amount compatible with the exclusion of adjacent binding sites, the electric dichroism decreased owing to the appearance of externally bound dye molecules with no contribution to the dichroism. Sonicated DNA was used to study the lengthening of the DNA molecule upon complexation. Although the viscosity of the complexes increased with the amount of binding, the rotational diffusion coefficient measured by the electric birefringence relaxation was not detectably affected. The absence of variation in the electric birefringence with the binding indicated that the DNA base stacking remained unaltered.     

Amine group of guanine enhances the binding of norfloxacin antibiotics to DNA.

The binding mode of norfloxacin, a quinolone antibacterial agent, in the synthetic polynucleotides poly[d(G-C)2], poly[d(I-C)2] and poly[d(A-T)2] was studied using polarized light spectroscopy, fluorescence spectroscopy and melting profiles. The absorption, circular and linear dichroism properties of norfloxacin are essentially the same for all the complexes, and the angle of electric transition dipole moment I and II of norfloxacin relative to the DNA helix axis is measured as 68-75 degrees for all complexes. These similarities indicate that the binding mode of norfloxacin is similar for all the polynucleotides. The decrease in the linear dichroism (LD) magnitude at 260 nm upon binding norfloxacin, which is strongest for the norfloxacin-poly[d(G-C)2] complex, and the identical melting temperature of poly[d(A-T)2] and poly[d(I-C)2] in the presence and absence of norfloxacin rule out the possibility of classic intercalation and minor groove binding. However, the characteristics of the fluorescence emission spectra of norfloxacin bound to poly[d(A-T)2] and to poly[d(I-C)2] are similar but are different to that of norfloxacin bound to poly[d(G-C)2]. As the amine group of the guanine base protrudes to the minor groove, this result strongly suggests that norfloxacin binds in the minor groove of B-form DNA in a nonclassic manner.     

Dissociation and unfolding of bovine odorant binding protein at acidic pH

Dissociation of bovine odorant binding protein (bOBP) dimers to monomers at pH 2.5 has been confirmed through size exclusion chromatography experiments. Moreover, structural and binding properties of the acidic monomer and neutral dimer have been compared using a combination of experimental (circular dichroism and fluorescence) and computational (molecular dynamics) techniques. The secondary and tertiary structures of bOBP are largely maintained at acidic pH, but molecular dynamics simulations suggest the loop regions (N-terminal residues, Omega-loop and C-terminal segments) are more relaxed and Phe36 and Tyr83 residues are involved in the regulation of the binding cavity entrance. The formation of a molten globule state at acidic pH, suggested by the strong enhancement of fluorescence of 8-anilino-1-naphtalenesulphonic acid (ANS), is not confirmed by any significant change in the near UV circular dichroism spectrum. Functionality measurements, deduced from the interaction of bOBP with 1-amino-anthracene (AMA), show that the binding capacity of the protein at acidic pH is preserved, though slightly looser than at neutral pH. Unfolding of acidic bOBP, induced by guanidinium chloride (GdnHCl), was investigated by means of CD spectroscopy, steady state fluorescence, fluorescence anisotropy and light scattering. The stability of the acidic monomer is lower than that of the neutral dimer, owing to the loss of the swapping interactions, but renaturation is completely reversible. Finally, in contrast with the neutral dimer, at low denaturant concentration some aggregation of the acidic monomer, which vanishes before the unfolding transition, has been observed.     

Insight into the binding of a non-toxic,self-assembling aromatic tripeptide with ct-DNA: Spectroscopic and viscositic studies

The report describes the synthesis, self-association and DNA binding studies of an aromatic tripeptide H-Phe-Phe-Phe-OH (FFF). The peptide backbone adopts β—sheet conformation both in solid and solution. In aqueous solution, FFF self-assembles to form nanostructured aggregates. Interactions of this peptide with calf-thymus DNA (ct-DNA) have been studied using various biophysical techniques including ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The value of mean binding constant calculated from UV and fluorescence spectroscopic data is (2.914 ± 0.74) x 10³ M^?1 which is consistent with an external binding mode. Fluorescence intercalator displacement (FID) assay, iodide quenching study, viscosity measurement and thermal denaturation study of DNA further confirm the groove binding mode of peptide, FFF with ct-DNA. MTT cell survival assay reveals very low cytotoxicity of the peptide toward human lung carcinoma cell line A549.     

竹红菌甲素敏化DNA的光氧化——甲素与DNA的结合及DNA某些理化性质变化

本文研究了光敏氧化前后甲素与DNA的结合以及这种结合对DNA-EB复合物荧光强度、DNA融解温度和圆二色谱的影响。利用Sephadex G-150凝胶过滤和同位素技术分离甲素-DNA复合物,测得光照前后甲素与DNA的结合率分别为15.2％和27.8％。荧光测定和琼脂糖凝胶电泳结果显示:甲素可阻碍DNA-EB复合物的形成、使DNA-EB复合物的荧光强度减弱,光照甲素对DNA-EB复合物的荧光淬灭作用较未光照者为强。甲素敏化DNA光氧化后的荧光淬灭、DNA融解温度降低和CD谱变化提示:甲素敏化DNA光氧化涉及碱基破坏及DNA双螺旋结构改变。     

Preparation, characterization, and DNA binding studies of water-soluble quercetin--molybdenum(VI) complex

DNA binding studies of flavonoids are needed to understand the reaction mechanism and improve drugs that target DNA. Quercetin (Q) is one of the most common flavonoids that can chelate metal ions and interact with double-stranded DNA. In the present work, UV absorption spectrophotometry, viscosimetry, circular dichroism, and fluorescence spectroscopic techniques were employed to study the interaction of water-soluble quercetin--molybdenum(VI) complex [Q-Mo(VI)] with calf thymus DNA. The binding constants (K(b)) for the complex with DNA were estimated to be 2.9?×?10(3) through spectroscopic titrations. Upon addition of the complex, significant decreases were observed in the viscosity of calf thymus DNA. Circular dichroic spectra indicated that there are certain detectable conformational changes in the DNA double helix when complex was added. Further, competitive methylene blue binding studies with fluorescence spectroscopy have shown that the complex can bind to DNA through nonintercalative mode. The experimental results suggest that Q-Mo(VI) binds to DNA via an outside binding mode.     

DNA binding studies of antibiotic drug cephalexin using spectroscopic and molecular docking techniques

The purpose of this study was to explore an accurate characterization of the binding interaction of antibiotic drug cephalexin with calf thymus DNA (CT-DNA) as a relevant biological target by using UV absorption, fluorescence spectroscopy and circular dichroism (CD) in vitro under simulated physiological conditions (pH = 7.4) and also through a molecular modeling study. The results showed that the drug interacts with the DNA helix via a minor groove binding mode. The thermodynamic parameters were calculated and showed that the reaction between the drug and CT-DNA was exothermic. In addition, the drug enforced traceable changes in the viscosity of DNA. The molecular modeling results indicated that cephalexin forcefully binds to the minor groove of DNA with a relative binding energy of ?21.02?kJ mol^?1. The obtained theoretical results were in good agreement with those obtained from experimental studies.