Mycobacterium tuberculosis cAMP receptor protein (Rv3676) differs from the Escherichia coli paradigm in its cAMP binding and DNA binding properties and transcription activation properties.

The pathogen Mycobacterium tuberculosis produces a burst of cAMP upon infection of macrophages. Bacterial cyclic AMP receptor proteins (CRP) are transcription factors that respond to cAMP by binding at target promoters when cAMP concentrations increase. Rv3676 (CRP(Mt)) is a CRP family protein that regulates expression of genes (rpfA and whiB1) that are potentially involved in M. tuberculosis persistence and/or emergence from the dormant state. Here, the CRP(Mt) homodimer is shown to bind two molecules of cAMP (one per protomer) at noninteracting sites. Furthermore, cAMP binding by CRP(Mt) was relatively weak, entropy driven, and resulted in a relatively small enhancement in DNA binding. Tandem CRP(Mt)-binding sites (CRP1 at -58.5 and CRP2 at -37.5) were identified at the whiB1 promoter (PwhiB1). In vitro transcription reactions showed that CRP1 is an activating site and that CRP2, which was only occupied in the presence of cAMP or at high CRP(Mt) concentrations in the absence of cAMP, is a repressing site. Binding of CRP(Mt) to CRP1 was not essential for open complex formation but was required for transcription activation. Thus, these data suggest that binding of CRP(Mt) to the PwhiB1 CRP1 site activates transcription at a step after open complex formation. In contrast, high cAMP concentrations allowed occupation of both CRP1 and CRP2 sites, resulting in inhibition of open complex formation. Thus, M. tuberculosis CRP has evolved several distinct characteristics, compared with the Escherichia coli CRP paradigm, to allow it to regulate gene expression against a background of high concentrations of cAMP.

Calorimetric and spectroscopic studies of Hoechst 33258: self-association and binding to non-cognate DNA.

Sequence and structure-specific molecular recognition of DNA by small molecules is an important goal in biophysical chemistry and drug discovery. Many candidate ligands possess flat aromatic surfaces and other molecular features that allow them to self-associate. In addition, non-specific binding to the target is a complicating feature of these interactions. Therefore, multiple equilibria are present and need to be accounted for in data analysis in order to obtain meaningful thermodynamic parameters. In order to address these issues we have systematically examined the bis-benzimidazole dye Hoechst 33258 (H33258) in terms of self-aggregation and binding to DNA oligonucleotides lacking any cognate minor groove A.T sites. This model system has been interrogated using isothermal titration calorimetry (ITC), circular dichroism (CD), fluorescence spectroscopy and pulsed gradient spin echo NMR. Three distinct binding events and ligand self-aggregation have been identified and, where possible, quantified. H33258 self-aggregation involves a step-wise aggregation mechanism, driven by stacking interactions. The DNA binding process includes two specific binding modes and non-specific DNA-templated H33258 stacking. We have written novel ITC data-fitting software (IC-ITC; freely available to the biophysics community), which simultaneously fits ligand aggregation and ligand-DNA binding. Here, this numerical analysis, which uses simulated annealing of complex calorimetric data representing multiple coupled equilibria, is described.

The dichotomy in the DNA-binding behaviour of ruthenium(II) complexes bearing benzoxazole and benzothiazole groups.

The substituted tris(bipyridine)ruthenium(II) complexes {[Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) [where bpy=2,2'-bipyridine and bbob=bis(benzoxazol-2-yl)-2,2'-bipyridine] have been prepared and compared to the previously studied complex [Ru(bpy)(2)(4,4'-bbtb)](2+) [where bbtb=bis(benzothiazol-2-yl)-2,2'-bipyridine]. From the UV/VIS titration studies, Delta-[Ru(bpy)(2)(4,4'-bbob)](2+) displays a stronger association than the Lambda-isomer with calf-thymus DNA (ct-DNA). For [Ru(bpy)(2)(5,5'-bbob)](2+), there appears to be minimal interaction with ct-DNA. The results of fluorescence titration studies suggest that [Ru(bpy)(2)(4,4'-bbob)](2+) gives an increase in emission intensity with increasing ct-DNA concentrations, with an enantiopreference for the Delta isomer, confirmed by membrane dialysis studies. The fluorescent intercalation displacement studies revealed that [Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) display a preference for more open DNA structures such as bulge and hairpin sequences. While Lambda-[Ru(bpy)(2)(4,4'-bbtb)](2+) has shown the most significant affinity for all the oligonucleotides sequences screened in previous studies, it is the Delta isomer of the comparable benzoxazole ruthenium(II) complex (Delta-[Ru(bpy)(2)(4,4'-bbob)](2+)) that preferentially binds to DNA.

Advances in the analysis of isothermal titration calorimetry data for ligand-DNA interactions.

Isothermal titration calorimetry (ITC) is a well established technique for the study of biological interactions. The strength of ITC is that it directly measures enthalpy changes associated with interactions. Experiments can also yield binding isotherms allowing quantification of equilibrium binding constants, hence an almost complete thermodynamic profile can be established. Principles and application of ITC have been well documented over recent years, experimentally the technique is simple to use and in ideal scenarios data analysis is trivial. However, ITC experiments can be designed such that previously inaccessible parameters can be evaluated. We outline some of these advances, including (1) exploiting different experimental conditions; (2) low affinity systems; (3) high affinity systems and displacement assays. In addition we ask the question: What if data cannot be fit using the fitting functions incorporated in the data-analysis software that came with your ITC? Examples where such data might be generated include systems following non 1:n binding patterns and systems where binding is coupled to other events such as ligand dissociation. Models dealing with such data are now appearing in literature and we summarise examples relevant for the study of ligand-DNA interactions.

7,8-Dihydropyrido[2,3-d]pyrimidin-2-one; a bicyclic cytosine analogue capable of enhanced stabilisation of DNA duplexes.

Incorporation of a bicyclic cytosine analogue, 3-beta-D-(2'-deoxyribofuranosyl)-7,8-dihydropyrido[2,3-d]pyrimidine, into synthetic DNA duplexes results in a greatly enhanced thermal stability (3-4 degrees C per modification) compared to the corresponding unmodified duplex.

Dinuclear monointercalating RuII complexes that display high affinity binding to duplex and quadruplex DNA.

The DNA duplex binding properties of previously reported dinuclear Ru(II) complexes based on the ditopic ligands tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2'',3''-j]phenazine (tppz) and tetraazatetrapyrido[3,2-a:2'3'-c:3'',2''-l:2''',3'''-n]pentacene (tatpp) are reported. Photophysical and biophysical studies indicate that, even at high ionic strengths, these complexes bind to duplex DNA, through intercalation, with affinities that are higher than any other monointercalating complex and are only equalled by DNA-threaded bisintercalating complexes. Additional studies at high ionic strengths using the 22-mer d(AG(3)[T(2)AG(3)](3)) [G3] human telomeric sequence reveal that the dinuclear tppz-based systems also bind with high affinity to quadruplex DNA. Furthermore, for these complexes, quadruplex binding is accompanied by a distinctive blue-shifted "light-switch" effect, characterized by higher emission enhancements than those observed in the analogous duplex effect. Calorimetry studies reveal that the thermodynamics of duplex and quadruplex binding is distinctly different, with the former being entirely entropically driven and the latter being both enthalpically and entropically favored.

Synthesis and characterization of biocompatible, thermoresponsive ABC and ABA triblock copolymer gelators.

The synthesis of doubly thermoresponsive PPO-PMPC-PNIPAM triblock copolymer gelators by atom transfer radical polymerization using a PPO-based macroinitiator is described. Provided that the PPO block is sufficiently long, dynamic light scattering and differential scanning calorimetry studies confirm the presence of two separate thermal transitions corresponding to micellization and gelation, as expected. However, these ABC-type triblock copolymers proved to be rather inefficient gelators: free-standing gels at 37 degrees C required a triblock copolymer concentration of around 20 wt%. This gelator performance should be compared with copolymer concentrations of 6-7 wt% required for the PNIPAM-PMPC-PNIPAM triblock copolymers reported previously. Clearly, the separation of micellar self-assembly from gel network formation does not lead to enhanced gelator efficiencies, at least for this particular system. Nevertheless, there are some features of interest in the present study. In particular, close inspection of the viscosity vs temperature plot obtained for a PPO43-PMPC160-PNIPAM81 triblock copolymer revealed a local minimum in viscosity. This is consistent with intramicelle collapse of the outer PNIPAM blocks prior to the development of the intermicelle hydrophobic interactions that are a prerequisite for macroscopic gelation.

Water-soluble organic dppz analogues--tuning DNA binding affinities, luminescence, and photo-redox properties.

Three new water-soluble dppz derivatives are reported, one of which binds to DNA with an affinity comparable to any mononuclear metal complex and also displays a high selectivity for GC sites.

DNA binding of an organic dppz-based intercalator.

An improved synthesis of a water-soluble derivative of dipyrido[3,2-a:2',3'-c]phenazine (dppz) is reported. The structures of both dppz and the cation ethylene-bipyridyldiylium-phenazine dinitrate [[1][(PF(6))(2)]] have been obtained via X-ray crystallography. Metal complex derivatives of dppz are very well studied. However, using the water soluble [1][(NO(3))(2)], the nature of the interaction of a simple dppz unit with duplex DNA has been investigated for the first time. In both organic solvents and water, 1 displays unstructured luminescence, assigned to an intramolecular charge transfer. The emission is quenched on binding to natural and synthetic duplex DNA, including poly(dA).poly(dT). A variety of techniques reveal that the cation binds to DNA with an affinity comparable to those of many metal dppz complexes, via an intercalative binding mode.

Expression and characterization of the two flavodoxin proteins of Bacillus subtilis, YkuN and YkuP: biophysical properties and interactions with cytochrome P450 BioI.

The two flavodoxins (YkuN and YkuP) from Bacillus subtilis have been cloned, overexpressed in Escherichia coli and purified. DNA sequencing, mass spectrometry, and flavin-binding properties showed that both YkuN and YkuP were typical short-chain flavodoxins (158 and 151 amino acids, respectively) and that an error in the published B. subtilis genome sequence had resulted in an altered reading frame and misassignment of YkuP as a long-chain flavodoxin. YkuN and YkuP were expressed in their blue (neutral semiquinone) forms and reoxidized to the quinone form during purification. Potentiometry confirmed the strong stabilization of the semiquinone form by both YkuN and YkuP (midpoint reduction potential for oxidized/semiquinone couple = -105 mV/-105 mV) with respect to the hydroquinone (midpoint reduction potential for semiquinone/hydroquinone couple = -382 mV/-377 mV). Apoflavodoxin forms were generated by trichloroacetic acid treatment. Circular dichroism studies indicated that flavin mononucleotide (FMN) binding led to considerable structural rearrangement for YkuP but not for YkuN. Both apoflavodoxins bound FMN but not riboflavin avidly, as expected for short-chain flavodoxins. Structural stability studies with the chaotrope guanidinium chloride revealed that there is moderate destabilization of secondary and tertiary structure on FMN removal from YkuN, but that YkuP apoflavodoxin has similar (or slightly higher) stability compared to the holoprotein. Differential scanning calorimetry reveals further differences in structural stability. YkuP has a lower melting temperature than YkuN, and its endotherm is composed of a single transition, while that for YkuN is biphasic. Optical and fluorimetric titrations with oxidized flavodoxins revealed strong affinity (K(d) values consistently <5 microM) for their potential redox partner P450 BioI, YkuN showing tighter binding. Stopped-flow reduction studies indicated that the maximal electron-transfer rate (k(red)) to fatty acid-bound P450 BioI occurs from YkuN and YkuP at approximately 2.5 s(-1), considerably faster than from E. coli flavodoxin. Steady-state turnover with YkuN or YkuP, fatty acid-bound P450 BioI, and E. coli NADPH-flavodoxin reductase indicated that both flavodoxins supported lipid hydroxylation by P450 BioI with turnover rates of up to approximately 100 min(-1) with lauric acid as substrate. Interprotein electron transfer is a likely rate-limiting step. YkuN and YkuP supported monohydroxylation of lauric acid and myristic acid, but secondary oxygenation of the primary product was observed with both palmitic acid and palmitoleic acid as substrates.

Thermodynamic and biophysical characterization of cytochrome P450 BioI from Bacillus subtilis.

Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an acyl bond cleavage reaction [Green, A. J., Rivers, S. L., Cheesman, M., Reid, G. A., Quaroni, L. G., Macdonald, I. D. G., Chapman, S. K., and Munro, A. W. (2001) J. Biol. Inorg. Chem. 6, 523-533. Stok, J. E., and De Voss, J. J. (2000) Arch. Biochem. Biophys. 384, 351-360]. Here we show novel binding features of P450 BioI--specifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (K(d) values in the range 0.1-160 microM). Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content). Steroids containing a carbonyl group at the C(3) position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI = -330 +/- 1 mV; for BioI as purified from E. coli (mixed-spin) = 228 +/- 2 mV; for palmitoleic acid-bound BioI = -199 +/- 2 mV). Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.

Roles of divalent metal ions in flap endonuclease-substrate interactions.

Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.

A facile route to bimetallic ruthenium dipyridophenazine complexes.

Using achiral coordinatively unsaturated metal complex building-blocks, the two step synthesis of a bimetallic complex containing independent [Ru(II)dppz] units tethered together by a linking 4,4'dipyridyl-1,5-pentane ligand is reported. Photophysical studies on this prototype system indicate that the characteristic luminescence of the [Ru(II)dppz] moieties is perturbed by self-quenching processes. Preliminary binding studies on the complex with natural and synthetic duplex DNA is reported. Luminescence and calorimetric titrations reveal that the complex does not show enhanced binding affinity with respect to analogous monometallic complexes. This result is interpreted by a consideration of the length and rigidity of the linker employed in the complex.

A ruthenium dipyridophenazine complex that binds preferentially to GC sequences.

Uniquely, a Ru11 complex of the dppz ligand shows a preference for GC sequences of DNA.

H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein.

H-NS plays a role in condensing DNA in the bacterial nucleoid. This 136 amino acid protein comprises two functional domains separated by a flexible linker. High order structures formed by the N-terminal oligomerization domain (residues 1-89) constitute the basis of a protein scaffold that binds DNA via the C-terminal domain. Deletion of residues 57-89 or 64-89 of the oligomerization domain precludes high order structure formation, yielding a discrete dimer. This dimerization event represents the initial event in the formation of high order structure. The dimers thus constitute the basic building block of the protein scaffold. The three-dimensional solution structure of one of these units (residues 1-57) has been determined. Activity of these structural units is demonstrated by a dominant negative effect on high order structure formation on addition to the full length protein. Truncated and site-directed mutant forms of the N-terminal domain of H-NS reveal how the dimeric unit self-associates in a head-to-tail manner and demonstrate the importance of secondary structure in this interaction to form high order structures. A model is presented for the structural basis for DNA packaging in bacterial cells.

Thermodynamics of drug-DNA interactions.

Many anticancer, antibiotic, and antiviral drugs exert their primary biological effects by reversibly interacting with nucleic acids. Therefore, these biomolecules represent a major target in drug development strategies designed to produce next generation therapeutics for diseases such as cancer. In order to improve the clinical efficacy of existing drugs and also to design new ones it is necessary to understand the molecular basis of drug-DNA interactions in structural, thermodynamic, and kinetic detail. The past decade has witnessed an increase in the number of rigorous biophysical studies of drug-DNA systems and considerable knowledge has been gained in the energetics of these binding reactions. This is, in part, due to the increased availability of high-sensitivity calorimetric techniques, which have allowed the thermodynamics of drug-DNA interactions to be probed directly and accurately. The focus of this article is to review thermodynamic approaches to examining drug-DNA recognition. Specifically, an overview of a recently developed method of analysis that dissects the binding free energy of these reactions into five component terms is presented. The results of applying this analysis to the DNA binding interactions of both minor groove drugs and intercalators are discussed. The solvent water plays a key role in nucleic acid structure and consequently in the binding of ligands to these biomolecules. Any rational approach to DNA-targeted drug design requires an understanding of how water participates in recognition and binding events. Recent studies examining hydration changes that accompany DNA binding by intercalators will be reviewed. Finally some aspects of cooperativity in drug-DNA interactions are described and the importance of considering cooperative effects when examining these reactions is highlighted.