Energetic basis of hydrogen bond formation in aqueous solution.

The thermodynamic forces driving the formation of H-bonds in macromolecules have long been the subject of speculation, theory and experiment. Comparison of the energetic parameters of AT and GC base pairs in DNA duplexes has recently led to the realisation that formation of a 'naked' hydrogen bond, i.e.

Thermodynamic basis of the α-helix and DNA duplex.

Analysis of calorimetric and crystallographic information shows that the α-helix is maintained not only by the hydrogen bonds between its polar peptide groups, as originally supposed, but also by van der Waals interactions between tightly packed apolar groups in the interior of the helix. These apolar contacts are responsible for about 60% of the forces stabilizing the folded conformation of the α-helix and their exposure to water on unfolding results in the observed heat capacity increment, i.e.

Forces maintaining the DNA double helix.

Despite the common acceptance that the enthalpy of DNA duplex unfolding does not depend on temperature and is greater for the CG base pair held by three hydrogen bonds than for the AT base pair held by only two, direct calorimetric measurements have shown that the enthalpic and entropic contributions of both base pairs are temperature dependent and at all temperatures are greater for the AT than the CG pair. The temperature dependence results from hydration of the apolar surfaces of bases that become exposed upon duplex dissociation. The larger enthalpic and entropic contributions of the AT pair are caused by water fixed by this pair in the minor groove of DNA and released on duplex dissociation.

Thermodynamics of DNA: heat capacity changes on duplex unfolding.

The heat capacity change, ΔCp, accompanying the folding/unfolding of macromolecules reflects their changing state of hydration. Thermal denaturation of the DNA duplex is characterized by an increase in ΔCp but of much lower magnitude than observed for proteins. To understand this difference, the changes in solvent accessible surface area (ΔASA) have been determined for unfolding the B-form DNA duplex into disordered single strands.

Forces maintaining the DNA double helix and its complexes with transcription factors.

Precise calorimetric studies of DNA duplexes of various length and composition have revised several long-held beliefs about the forces holding together the double helix and its complexes with the DNA binding domains (DBDs) of transcription factors. Heating DNA results in an initial non-cooperative increase of torsional oscillations in the duplex, leading to cooperative dissociation of its strands accompanied by extensive heat absorption and a significant heat capacity increment. The enthalpy and entropy of duplex dissociation are therefore temperature dependent quantities.

Translational Entropy and DNA Duplex Stability.

Investigation of folding/unfolding DNA duplexes of various size and composition by superprecise calorimetry has revised several long-held beliefs concerning the forces responsible for the formation of the double helix. It was established that: 1) the enthalpy and the entropy of duplex unfolding are temperature dependent, increasing with temperature rise and having the same heat capacity increment for CG and AT pairs; 2) the enthalpy of AT melting is greater than that of the CG pair, so the stabilizing effect of the CG pair in comparison with AT results not from its larger enthalpic contribution (as expected from its extra hydrogen bond), but from the larger entropic contribution of the AT pair that results from its ability to fix ordered water in the minor groove and release it upon duplex unfolding; 3) the translation entropy, resulting from the appearance of a new kinetic unit on duplex dissociation, determines the dependence of duplex stability on its length and its concentration (it is an order-of-magnitude smaller than predicted from the statistical mechanics of gases and is fully expressed by the stoichiometric correction term); 4) changes in duplex stability on reshuffling the sequence (the "nearest-neighbor effect") result from the immobilized water molecules fixed by AT pairs in the minor groove; and 5) the evaluated thermodynamic components permit a quantitative expression of DNA duplex stability.

Role of water in the formation of macromolecular structures.

This review shows that water in biological systems is not just a passive liquid solvent but also a partner in the formation of the structure of proteins, nucleic acids and their complexes, thereby contributing to the stability and flexibility required for their proper function. Reciprocally, biological macromolecules affect the state of the water contacting them, so that it is only partly in the normal liquid state, being somewhat ordered when bound to macromolecules. While the compaction of globular proteins results from the reluctance of their hydrophobic groups to interact with water, the collagen superhelix is maintained by water forming a hydroxyproline-controlled frame around this coiled-coil macromolecule.

The energetic basis of the DNA double helix: a combined microcalorimetric approach.

Microcalorimetric studies of DNA duplexes and their component single strands showed that association enthalpies of unfolded complementary strands into completely folded duplexes increase linearly with temperature and do not depend on salt concentration, i.e. duplex formation results in a constant heat capacity decrement, identical for CG and AT pairs.

The linker of the interferon response factor 3 transcription factor is not unfolded.

Interferon response factor 3 (IRF-3) is a transcription factor that plays an essential role in controlling the synthesis of interferon-β (IFN-β) and is a protein consisting of two well-defined domains, the N-terminal DNA-binding and the C-terminal dimerization domains, connected by a 75-residue linker, supposedly unfolded. However, it was not clear whether in intact IRF-3 this linker segment of the chain, which carries the nuclear export signal and includes a region of high helical propensity, remains unfolded. This has been investigated using nuclear magnetic resonance by ligating the (15)N-labeled linker to the unlabeled N-terminal and C-terminal domains.

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.

Unfolding of bZIP dimers formed by the ATF-2 and c-Jun transcription factors is not a simple two-state transition.

The varied selectivity of bZIP transcription factors stems from the fact that they are dimers consisting of two not necessarily identical subunits held together by a leucine zipper dimerization domain. Determining their stability is therefore important for understanding the mechanism of formation of these transcription factors. The most widely used approach for this problem consists of observing temperature-induced dissociation of the bZIPs by any means sensitive to their structural changes, particularly optical methods.

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).

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.

Microcalorimetry of proteins and their complexes.

Ultrasensitive microcalorimetric techniques for measuring the heat capacities of proteins in dilute solutions over a broad temperature range (DSC) and the heats of protein reactions at fixed temperatures (ITC) are described and the methods of working with these instruments are considered. Particular attention is paid to analyzing the thermal properties of individual proteins, their stability, the energetics of their folding, and their association with specific macromolecular partners. Use of these calorimetric methods is illustrated with examples of small compact globular proteins, small proteins having loose noncompact structure, multidomain proteins, and protein complexes, particularly with DNA.

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.

Assembling the human IFN-beta 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.

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.

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-beta enhancer have been studied.

DNA bending by charged peptides: electrophoretic and spectroscopic analyses.

We are testing the idea that placement of fixed charges near one face of the DNA double helix can induce DNA bending by a purely electrostatic mechanism. If stretching forces between DNA phosphates are significant, fixed charges should induce DNA bending by asymmetrically modulating these forces. We have previously tested this hypothesis by adding charged residues to small bZIP DNA binding peptides and monitoring DNA bending using electrophoretic phasing assays.

What drives proteins into the major or minor grooves of DNA?

The energetic profiles of a significant number of protein-DNA systems at 20 degrees C reveal that, despite comparable Gibbs free energies, association with the major groove is primarily an enthalpy-driven process, whereas binding to the minor groove is characterized by an unfavorable enthalpy that is compensated by favorable entropic contributions. These distinct energetic signatures for major versus minor groove binding are irrespective of the magnitude of DNA bending and/or the extent of binding-induced protein refolding. The primary determinants of their different energetic profiles appear to be the distinct hydration properties of the major and minor grooves; namely, that the water in the A+T-rich minor groove is in a highly ordered state and its removal results in a substantial positive contribution to the binding entropy.

The extended arms of DNA-binding domains: a tale of tails.

DNA-binding domains (DBDs) frequently have N- or C-terminal tails, rich in lysine and/or arginine and disordered in free solution, that bind the DNA separately from and in the opposite groove to the folded domain. Is their role simply to increase affinity for DNA or do they have a role in specificity, that is, sequence recognition? One approach to answering this question is to analyze the contribution of such tails to the overall energetics of binding. It turns out that, despite similarities of amino acid sequence, three distinct categories of DBD extension exist: (i) those that are purely electrostatic and lack specificity, (ii) those that are largely non-electrostatic with a high contribution to specificity and (iii) those of mixed character that show sequence preference.

Microcalorimetry of biological macromolecules.

The capabilities of contemporary differential scanning and isothermal titration microcalorimetry for studying the thermodynamics of protein unfolding/refolding and their association with partners, particularly target DNA duplexes, are considered. It is shown that the predenaturational changes of proteins must not be ignored in studying the thermodynamics of formation of their native structure and their complexes with partners, particularly their cognate DNA duplexes.

Forces driving the binding of homeodomains to DNA.

Homeodomains are helix-turn-helix type DNA-binding domains that exhibit sequence-specific DNA binding by insertion of their "recognition" alpha helices into the major groove and a short N-terminal arm into the adjacent minor groove without inducing substantial distortion of the DNA. The stability and DNA binding of four representatives of this family, MATalpha2, engrailed, Antennapedia, and NK-2, and truncated forms of the last two lacking their N-terminal arms have been studied by a combination of optical and microcalorimetric methods at different temperatures and salt concentrations. It was found that the stability of the free homeodomains in solution is rather low and, surprisingly, is reduced by the presence of the N-terminal arm for the Antennapedia and NK-2 domains.