Anatoly I. Dragan

Interpreting protein/DNA interactions: distinguishing specific from non-specific and electrostatic from non-electrostatic components

We discuss the effectiveness of existing methods for understanding the forces driving the formation of specific protein–DNA complexes. Theoretical approaches using the Poisson–Boltzmann (PB) equation to analyse interactions between these highly charged macromolecules to form known structures are contrasted with an empirical approach that analyses the effects of salt on the stability of these complexes and assumes that release of counter-ions associated with the free DNA plays the dominant role in their formation. According to this counter-ion condensation (CC) concept, the salt-dependent part of the Gibbs energy of binding, which is defined as the electrostatic component, is fully entropic and its dependence on the salt concentration represents the number of ionic contacts present in the complex. It is shown that although this electrostatic component provides the majority of the Gibbs energy of complex formation and does not depend on the DNA sequence, the salt-independent part of the Gibbs energy—usually regarded as non-electrostatic—is sequence specific. The CC approach thus has considerable practical value for studying protein/DNA complexes, while practical applications of PB analysis have yet to demonstrate their merit.

Unfolding of a Leucine zipper is not a Simple Two-state Transition

Temperature-induced unfolding of the leucine zipper, an alpha-helical, double-stranded, coiled-coil, was studied by circular dichroism spectroscopy, spectrofluorimetry and heat capacity scanning calorimetry. It is shown that this process does not represent a simple two-state transition, as previously believed, but consists of several stages. The first transition starts at the very beginning of heating from 0 degrees C and proceeds with significant heat absorption and decrease of ellipticity. This transition does not depend on the concentration of protein and is sensitive to modification of the N terminus; it is therefore associated with unfolding or fraying of this part of the leucine zipper. The second transition takes place at a considerably higher temperature; it is more pronounced than the first one and does not depend on the concentration of protein, i.e. it is unimolecular. This transition is sensitive to modification of both termini of the leucine zipper and affects the optical properties of a tryptophan residue placed in the central part of the zipper. It therefore involves the whole dimer but does not result in its dissociation, presumably being associated with some repacking of the coiled-coil. This second transition is followed at higher temperatures by the concentration-dependent cooperative unfolding/dissociation of the two strands. The enthalpy and entropy of the temperature-induced structural changes of the leucine zipper that take place before its cooperative unfolding/dissociation comprises almost 40% of the total enthalpy and entropy of unfolding of the completely folded coiled-coil, the state in which it appears to be below 0 degrees C. Comparison of the total enthalpy of leucine zipper unfolding with that of a single-stranded alpha-helix shows that their temperature-dependence correlates with the extent of intramolecular non-polar contacts and allows an assessment of the enthalpy of hydrogen bonding in alpha-helices, which appears to be about 3.3kJmol(-1) at 20 degrees C.

DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, whilst the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0 degrees C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, whilst binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box.

Mechanisms of activation of interferon regulator factor 3: the role of C-terminal domain phosphorylation in IRF-3 dimerization and DNA binding

The interferon regulatory transcription factor (IRF-3) is activated by phosphorylation of Ser/Thr residues clustered in its C-terminal domain. Phosphorylation of these residues, which increases the negative charge of IRF-3, results in its dimerization and association with DNA, despite the increase in repulsive electrostatic interactions. To investigate this surprising effect, the dimerization of IRF-3 and two phosphomimetic mutants, 2D (S396D, S398D) and 5D (S396D, S398D, S402D, T404D and S405D), and their binding to single-site PRDI and double-site PRDIII–PRDI DNA sequences from the IFN-β enhancer have been studied. It was found that: (a) the mutations in the C-terminal domain do not affect the state of the DNA-binding N-terminal domain or its ability to bind target DNA; (b) in the 5D-mutant, the local increase of negative charge in the C-terminal domain induces restructuring, resulting in the formation of a stable dimer; (c) dimerization of IRF-3 is the basis of its strong binding to PRDIII–PRDI sites since binding of 5D to the single PRDI site is similar to that of inactivated IRF-3. Analysis of the binding characteristics leads to the conclusion that binding of dimeric IRF-3 to the DNA with two tandem-binding sites, which are twisted by ∼100° relative to each other, requires considerable work to untwist and/or bend the DNA.

The cost of DNA bending

Experimental data on protein-DNA interactions highlight a surprising peculiarity of protein binding to the minor groove: in contrast to major groove binding, which proceeds with heat release and does not induce substantial deformation of DNA, minor groove binding takes place at AT-rich sites, proceeds with heat absorption and results in significant DNA bending. By forming a highly ordered and dense spine in the minor groove of AT-rich DNA, water plays an essential role in defining the energetic signature of protein-minor groove binding. Removal of this water requires minimal work and results in significant loss of rigidity in the DNA, which can then easily acquire the conformation imposed by the bound protein. Therefore the introduction of substantial bends into the DNA is not energetically expensive.

Assembling the Human IFN-β Enhanceosome in Solution

Assembly of interferon-beta enhanceosome from its individual protein components and of enhancer DNA has been studied in solution using a combination of fluorescence anisotropy, microcalorimetry, and CD titration. It was shown that the enhancer binds only one full-length phosphomimetic IRF-3 dimer at the PRDIII-PRDI sites, and this binding does not exhibit cooperativity with binding of the ATF-2/c-Jun bZIP (leucine zipper dimer with basic DNA recognition segments) heterodimer at the PRDIV site. The orientation of the bZIP pair is, therefore, not determined by the presence of the IRF-3 dimer, but is predetermined by the asymmetry of the PRDIV site. In contrast, bound IRF-3 dimer interacts strongly with the NF-kappaB (p50/p65) heterodimer bound at the neighboring PRDII site. The orientation of bound NF-kappaB is also predetermined by the asymmetry of the PRDII site and is the opposite of that found in the crystal structure. The HMG-I/Y protein, proposed as orchestrating enhanceosome assembly, interacts specifically with the PRDII site of the interferon-beta enhancer by inserting its DNA-binding segments (AT hooks) into the minor groove, resulting in a significant increase in NF-kappaB binding affinity for the major groove of this site.

Stability and DNA-Binding Ability of the bZIP Dimers Formed by the ATF-2 and c-Jun Transcription Factors

The dimer formed by the ATF-2 and c-Jun transcription factors is one of the main components of the human interferon-beta enhanceosome. Although these two transcription factors are able to form two homodimers and one heterodimer, it is mainly the heterodimer that participates in the formation of this enhanceosome, binding specifically to the positive regulatory domain IV (PRDIV) site of the enhancer DNA. To understand this surprising advantage of the heterodimer, we investigated the association of these transcription factors using fragments containing the basic DNA-recognition segment and the basic leucine zipper domain (bZIP). It was found that the probability of forming the hetero-bZIP significantly exceeds the probability of forming homo-bZIPs, and that the hetero-bZIP interacts more strongly with the PRDIV site of the interferon-beta enhancer, especially in the orientation that places the folded ATF-2 basic segment in the upstream half of this asymmetric site. The effect of salt on the formation of the ATF-2/c-Jun dimer and on its ability to bind the target PRDIV site showed that electrostatic interactions between the charged groups of these proteins and with DNA play an essential role in the formation of the asymmetric ATF-2/c-Jun/PRDIV complex.

Use of Fluorescence Resonance Energy Transfer (FRET) in Studying Protein-induced DNA Bending

The specific association of many DNA-binding proteins with DNA frequently results in significant deformation of the DNA. Protein-induced DNA bends depend on the protein, the DNA sequence, the environmental conditions, and in some cases are very substantial, implying that DNA bending has important functional significance. The precise determination of the DNA deformation caused by proteins under various conditions is therefore of importance for understanding the biological role of the association. This review considers methods for the investigation of protein-induced DNA bending by measuring the change in fluorescence resonance energy transfer (FRET) between fluorophores placed at the ends of the target DNA duplex. This FRET technique is particularly efficient when the protein-induced bend in the DNA is considerable and results in a significant decrease in the distance between the DNA ends bearing the fluorophores. However, in the case of small bends the change of distance between the ends of short DNA duplexes, as typically used in protein binding experiments (about 16-20 bp), is too small to be detected accurately by FRET. In such cases the change of the distance between the fluorophores can be increased by using levers attached to the binding site, that is, using two bulges to construct a U-shaped DNA in which the central part contains the protein-binding site and the fluorophores are attached to the ends of the perpendicularly directed arms.

DNA hydration studied by pressure perturbation scanning microcalorimetry.

Pressure perturbation differential scanning calorimetry was used to determine thermal expansion coefficients and thus temperature-induced volume changes of DNA duplexes differing in their GC/AT content. It was shown that the temperature-induced unfolding of the DNA duplexes proceeds with a significant increase of the thermal expansion coefficient and the partial volume of the DNA. Unusually, large temperature-induced changes in the partial volume were observed for an AT-rich dodecamer, a finding consistent with previous crystallographic studies showing the presence of highly ordered water molecules hydrating the minor groove of such duplexes. The data show that the density of this ordered water is substantially higher than that of the bulk water. This ordered water cannot, therefore, be equated to ice at normal pressures but it thermodynamically resembles ice formed at high pressures.

Metal-enhanced PicoGreen fluorescence: application to fast and ultra-sensitive pg/ml DNA quantitation.

In this paper we provide both a theoretical and experimental analysis of the sensitivity of a DNA quantitation assay using a fluorescent chromophore which non-covalently binds dsDNA. It is well-known that the range of DNA concentrations available for fluorescence quantitation depends on the concentration of the chromophore, its affinity for nucleic acids, the binding site size on DNA and the ratio between the fluorescence intensity of the chromophore when bound to DNA compared to free chromophore in solution. We present experimental data obtained for a PicoGreen (PG)/DNA quantitation assay, which is in complete agreement with the results of our theoretical analysis. Experimentally measured PG-fluorescence intensity vs DNA concentration functions were fitted by a derived analytical expression, in which parameters of PG binding to DNA and chromophore fluorescence properties were included. We show that silver nanoparticles significantly increase the ratio between the fluorescence of PG bound to DNA and free PG, due to the metal-enhanced fluorescence effect (MEF), which enhances the lower limit of detectability of DNA concentrations by several orders of magnitude. An additional order of magnitude increase of PG/DNA assay sensitivity (~1 pg/ml) can be achieved by decreasing the PG concentration. We show herein that the use of MEF substrates in surface assays has a profound effect on assay sensitivity.