RESUMEN
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. without other accompanying Van der Waals close contacts, is a non-enthalpic process driven by the entropy increase resulting from release of tightly bound water molecules from the component polar groups. This unexpected conclusion finds a parallel in the formation of ionic bonds, for example between the amino groups of DNA binding proteins and the oxygens of DNA phosphate groups that are also non-enthalpic and entropy driven. The thermodynamic correspondence between these two types of polar non-covalent bonding implies that the non-enthalpic nature of base pairing in DNA is not particular to that specific structural circumstance.
Asunto(s)
ADN , Agua , Emparejamiento Base , ADN/química , Enlace de Hidrógeno , TermodinámicaRESUMEN
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. Analysis of the experimental thermodynamic characteristics of unfolding/refolding DNA duplexes of various compositions shows that the enthalpy of base pairing is negligibly small, while the entropic contribution is considerable. Thus, DNA base pairing is entropy driven and is coupled to the enthalpy driven van der Waals base pair stacking. Each of these two processes is responsible for about half the Gibbs energy of duplex stabilization, but all the enthalpy, i.e., the total heat of melting, results from dissociation of the stacked base pairs. Both these processes tightly cooperate: while the pairing of conjugate bases is critical for recognition of complementary strands, stacking of the flat apolar surfaces of the base pairs reinforces the DNA duplex formed.
Asunto(s)
ADN/química , Fenómenos Mecánicos , Emparejamiento Base , Fenómenos Biomecánicos , Propiedades de Superficie , Termodinámica , Agua/químicaRESUMEN
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. These showed that the polar component represents ~ 55% of the total increase in ASA, in contrast to globular proteins of similar molecular weight for which the polar component is only about 1/3rd of the total. As the exposure of polar surface results in a decrease of ΔCp, this explains the much reduced heat capacity increase observed for DNA and emphasizes the enhanced role of polar interactions in maintaining duplex structure. Appreciation of a non-zero ΔCp for DNA has important consequences for the calculation of duplex melting temperatures (Tm). A modified approach to Tm prediction is required and comparison is made of current methods with an alternative protocol.
Asunto(s)
ADN/química , Calor , Secuencia de Bases , ADN/genética , Desnaturalización de Ácido Nucleico , Propiedades de Superficie , TermodinámicaRESUMEN
The nature of water on the surface of a macromolecule is reflected in the temperature dependence of the heat effect, i.e., the heat capacity change, ΔCp, that accompanies its removal on forming a complex. The relationship between ΔCp and the nature of the surface dehydrated cannot be modeled for DNA by the use of small molecules, as previously done for proteins, since the contiguous surfaces of the grooves cannot be treated as the sum of small component molecules such as nucleotides. An alternative approach is used here in which ΔCp is measured for the formation of several protein/DNA complexes and the calculated contribution from protein dehydration subtracted to yield the heat capacity change attributable to dehydration of the DNA. The polar and apolar surface areas of the DNA dehydrated on complex formation were calculated from the known structures of the complexes, allowing heat capacity coefficients to be derived representing dehydration of unit surface area of polar and apolar surface in both grooves. Dehydration of apolar surfaces in both grooves is essentially identical and accompanied by a reduction in ΔCp by about 3 J K-1 mol-1 (Å2)-1, a value of somewhat greater magnitude than observed for proteins {ΔCp = - 1.79 J K-1 mol-1 (Å2)-1}. In contrast, dehydration of polar surfaces is very different in the two grooves: in the minor groove ΔCp increases by 2.7 J K-1 mol-1 (Å2)-1, but in the major groove, although ΔCp is also positive, it is low in value: + 0.4 J K-1 mol-1 (Å2)-1. Physical explanations for the magnitudes of ΔCp are discussed.
Asunto(s)
ADN/química , Calor , Conformación de Ácido Nucleico , Secuencia de Bases , ADN/genética , Modelos Moleculares , Propiedades de Superficie , Agua/químicaRESUMEN
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.
Asunto(s)
ADN/química , Entropía , Emparejamiento Base , Secuencia de Bases , ADN/genéticaRESUMEN
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. As for DNA, its stability and rigidity are linked to water fixed by AT pairs in the minor groove: this leads to the enthalpic contribution of AT pairs exceeding that of GC pairs, but this is overbalanced by their greater entropy contribution, with the result that AT pairs melt at lower temperatures than GCs. Loss of this water drives transcription factor binding to the minor groove.
Asunto(s)
Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Agua/metabolismo , Animales , Colágeno/química , Colágeno/metabolismo , ADN/química , ADN/metabolismo , Humanos , Agua/químicaRESUMEN
Structural modifications to interacting systems frequently lead to changes in both the enthalpy (heat) and entropy of the process that compensate each other, so that the Gibbs free energy is little changed: a major barrier to the development of lead compounds in drug discovery. The conventional explanation for such enthalpy-entropy compensation (EEC) is that tighter contacts lead to a more negative enthalpy but increased molecular constraints, i.e., a compensating conformational entropy reduction. Changes in solvation can also contribute to EEC but this contribution is infrequently discussed. We review long-established and recent cases of EEC and conclude that the large fluctuations in enthalpy and entropy observed are too great to be a result of only conformational changes and must result, to a considerable degree, from variations in the amounts of water immobilized or released on forming complexes. Two systems exhibiting EEC show a correlation between calorimetric entropies and local mobilities, interpreted to mean conformational control of the binding entropy/free energy. However, a substantial contribution from solvation gives the same effect, as a consequence of a structural link between the amount of bound water and the protein flexibility. Only by assuming substantial changes in solvation-an intrinsically compensatory process-can a more complete understanding of EEC be obtained. Faced with such large, and compensating, changes in the enthalpies and entropies of binding, the best approach to engineering elevated affinities must be through the addition of ionic links, as they generate increased entropy without affecting the enthalpy.
Asunto(s)
Descubrimiento de Drogas/métodos , Entropía , Calor , Solventes/química , Humanos , Ligandos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismoRESUMEN
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. Although duplex thermostability increases with CG content, the enthalpic and entropic contributions of an AT pair to duplex formation exceed that of a CG pair when compared at the same temperature. The reduced contribution of AT pairs to duplex stabilization comes not from their lower enthalpy, as previously supposed, but from their larger entropy contribution. This larger enthalpy and particularly the greater entropy results from water fixed by the AT pair in the minor groove. As the increased entropy of an AT pair exceeds that of melting ice, the water molecule fixed by this pair must affect those of its neighbors. Water in the minor groove is, thus, orchestrated by the arrangement of AT groups, i.e. is context dependent. In contrast, water hydrating exposed nonpolar surfaces of bases is responsible for the heat capacity increment on dissociation and, therefore, for the temperature dependence of all thermodynamic characteristics of the double helix.
Asunto(s)
ADN/química , Termodinámica , Emparejamiento Base , Calorimetría , Rastreo Diferencial de Calorimetría , Conformación de Ácido Nucleico , Desnaturalización de Ácido NucleicoRESUMEN
A simple, efficient, and reliable method is demonstrated for cloning long tandem arrays of the 601 nucleosomal positioning sequence. In addition, it is shown that such long arrays can be ligated together in vitro with high efficiency. By combining these two procedures it becomes straightforward to synthesize customized arrays that contain different (or variable) nucleosomal repeat lengths (NRLs) and monosome units bearing chemical modifications such as fluorophores, methyl groups, and reaction sites. This is, therefore, an enabling technology for the in vitro study of chromatin structure and function.
Asunto(s)
Nucleosomas/genética , Clonación MolecularRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/química , Animales , ADN/metabolismo , Humanos , Conformación de Ácido Nucleico , Termodinámica , AguaRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Entropía , Unión Proteica , Electricidad EstáticaRESUMEN
The UBTF E210K neuroregression syndrome is a predominantly neurological disorder caused by recurrent de novo dominant variants in Upstream Binding Factor, that is, essential for transcription of the ribosomal RNA genes. This unusual form of ribosomopathy is characterized by a slow decline in cognition, behavior, and sensorimotor functioning during the critical period of development. UBTF (or UBF) is a multi-HMGB-box protein that acts both as an epigenetic factor to establish "open" chromatin on the ribosomal genes and as a basal transcription factor in their RNA Polymerase I transcription. Here we review the possible mechanistic connections between the UBTF variants, ribosomal RNA gene transcription and the neuroregression syndrome, and suggest that DNA topology may play an important role.
RESUMEN
Interferon response factor 3 (IRF-3) is a transcription factor that plays an essential role in controlling the synthesis of interferon-ß (IFN-ß) 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. It was found that, while the linker alone is indeed in a completely unfolded state, when ligated to the C-terminal domain it shows some ordering, and this ordering becomes much more pronounced when the linker is also ligated to the N-terminal domain. Thus, in intact IRF-3, the linker represents a folded structural domain; i.e., IRF-3 is a three-domain globular protein. Light scattering studies of wild-type IRF-3 showed that these three domains are tightly packed, and therefore, the dimer of IRF-3, which is formed upon phosphorylation of its C-terminal domains following virus invasion, must be a rather rigid and compact construction. One would then expect that binding of such a dimer to its tandem recognition sites PRDIII and PRDI, which are located on opposing faces of the IFN-ß enhancer DNA, should result in deformation of the DNA. Analysis of the characteristics of binding of the monomeric and dimeric IRF-3 to the enhancer DNA indeed showed that formation of this complex requires considerable work for deformation of its components, most likely bending of the DNA. Such bending was confirmed by atomic force microscopy of dimeric IRF-3 bound to the PRDII-PRDI tandem recognition sites placed at the middle of a 300 bp DNA probe. Bending of DNA by IRF-3 must be significant in the assembly and function of the IFN-ß enhancer.
Asunto(s)
Factor 3 Regulador del Interferón/química , ADN/química , Factor 3 Regulador del Interferón/genética , Microscopía de Fuerza Atómica , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Desplegamiento ProteicoRESUMEN
Antibodies to the six chicken histone H1 subtypes and the variant histone H5 have been used in immunoprecipitations of crosslinked chromatin fragments (xChIPs) to map linker histones across the ß-globin locus and the widely expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and carbonic anhydrase (CA) genes in three cell types: 15-day embryo chicken erythrocytes, 15-day embryo chicken brain and the early erythroid cell line HD24. In erythrocytes, where the ß-adult and ß-hatching genes are active, the H1.01, H1.11L and H1.11R subtypes are substantially depleted throughout the ß-globin locus and the neighboring heterochromatin, in contrast to the other four subtypes, in particular the more abundant H5. Active genes therefore carry high levels of some but not all linker histone subtypes. The situation is similar in HD24 cells, except that substantial depletions are found at the promoters of the adult ß(A) and embryonic ß(ρ) and ß(ε) genes, despite these genes not yet being active in HD24 cells. The distributions in the brain tissue are characterised by the absence of H1.02, H1.03 and H5 from the hypersensitive site HS3 and from the ß-adult 3' enhancer for the H1.11L and H1.11R subtypes. The data show that although linker histone subtypes play distinct cell-type specific roles in gene regulation, their widespread distribution indicates they are not intrinsically inhibitory to basic chromatin transactions.
Asunto(s)
Histonas/genética , Histonas/metabolismo , Proteínas Represoras/metabolismo , Animales , Embrión de Pollo , Inmunoprecipitación de Cromatina , Mapeo Cromosómico/métodos , Reactivos de Enlaces Cruzados/farmacología , Regulación de la Expresión Génica , Sitios Genéticos , Humanos , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/fisiología , Proteínas Represoras/clasificación , Proteínas Represoras/inmunología , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
Lysine residues within histones can be mono-, di - or tri-methylated. In Saccharomyces cerevisiae tri-methylation of Lys 4 of histone H3 (K4/H3) correlates with transcriptional activity, but little is known about this methylation state in higher eukaryotes. Here, we examine the K4/H3 methylation pattern at the promoter and transcribed region of metazoan genes. We analysed chicken genes that are developmentally regulated, constitutively active or inactive. We found that the pattern of K4/H3 methylation shows similarities to S. cerevisiae. Tri-methyl K4/H3 peaks in the 5' transcribed region and active genes can be discriminated by high levels of tri-methyl K4/H3 compared with inactive genes. However, our results also identify clear differences compared to yeast, as significant levels of K4/H3 methylation are present on inactive genes within the beta-globin locus, implicating this modification in maintaining a 'poised' chromatin state. In addition, K4/H3 di-methylation is not genome-wide and di-methylation is not uniformly distributed throughout the transcribed region. These results indicate that in metazoa, di- and tri-methylation of K4/H3 is linked to active transcription and that significant differences exist in the genome-wide methylation pattern as compared with S. cerevisiae.
Asunto(s)
Células Eucariotas/metabolismo , Regulación de la Expresión Génica/genética , Histonas/metabolismo , Lisina/metabolismo , Metilación , Animales , Pollos , Mapeo Cromosómico , Genes/genética , Genoma , Globinas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Activación Transcripcional/genéticaRESUMEN
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. Because in all cases the tails also increase the affinity for target DNA, they represent a crucial component of the machinery for selective gene activation or repression.
Asunto(s)
Proteínas de Unión al ADN/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , TermodinámicaRESUMEN
Understanding the forces driving formation of protein/DNA complexes requires measurement of the Gibbs energy of association, DeltaG, and its component enthalpic, DeltaH, and entropic, DeltaS, contributions. Isothermal titration calorimetry provides the enthalpy (heat) of the binding reaction and an estimate of the association constant, if not too high. Repeating the ITC experiment at several temperatures yields DeltaC ( p ), the change in heat capacity, an important quantity permitting extrapolation of enthalpies and entropies to temperatures outside the experimental range. Binding constants, i.e. Gibbs energies, are best obtained by optical methods such as fluorescence at temperatures where the components are maximally folded. Since DNA-binding domains are often partially unfolded at physiological temperatures, the ITC-observed enthalpy of binding may need to be corrected for the negative contribution from protein refolding. This correction is obtained by differential scanning calorimetric melting of the free DNA-binding domain. Corrected enthalpies are finally combined with accurate Gibbs energies to yield the entropy factor (TDeltaS) at various temperatures. Gibbs energies can be separated into electrostatic and non-electrostatic contributions from the ionic strength dependence of the binding constant.
Asunto(s)
Calorimetría/métodos , ADN/metabolismo , Proteínas/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Polarización de Fluorescencia , Humanos , Indicadores y Reactivos , Ratones , Pliegue de Proteína , Soluciones , Electricidad Estática , Termodinámica , VolumetríaRESUMEN
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. Since the entropic forces driving protein binding into the minor groove are a consequence of displacing water ordered by the regular arrangement of polar contacts, they cannot be regarded as hydrophobic.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Composición de Base , Sitios de Unión , Entropía , Modelos Químicos , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Pliegue de Proteína , Electricidad Estática , TermodinámicaRESUMEN
Native chromatin IP assays were used to define changes in core histone acetylation at the lysozyme locus during developmental maturation of chicken macrophages and stimulation to high-level expression by lipo-polysaccharide. In pluripotent precursors the lysozyme gene (Lys) is inactive and there is no acetylation of core histones at the gene, its promoter or at the upstream cis-control elements. In myeloblasts, where there is a very low level of Lys expression, H4 acetylation appears at the cis-control elements but not at the Lys gene or its promoter: neither H3 nor H2B become significantly acetylated in myeloblasts. In mature macrophages, Lys expression increases 5-fold: H4, H2B and H2A.Z are all acetylated at the cis-control elements but H3 remains unacetylated except at the -2.4 S silencer. Stimulation with LPS increases Lys expression a further 10-fold: this is accompanied by a rise in H3 acetylation throughout the cis-control elements; H4 and H2B acetylation remain substantial but acetylation at the Lys gene and its promoter remains low. Acetylation is thus concentrated at the cis-control elements, not at the Lys gene or its immediate promoter. H4 acetylation precedes H3 acetylation during development and H3 acetylation is most directly linked to high-level Lys expression.
Asunto(s)
Pollos/genética , Elementos de Facilitación Genéticos , Histonas/metabolismo , Macrófagos/metabolismo , Muramidasa/genética , Acetilación , Animales , Anticuerpos , Línea Celular , Pollos/crecimiento & desarrollo , Pollos/metabolismo , Inmunoprecipitación de Cromatina , Regulación del Desarrollo de la Expresión Génica , Células Precursoras de Granulocitos/metabolismo , Histonas/inmunología , Muramidasa/metabolismo , Células Madre Pluripotentes/metabolismo , ARN Mensajero/metabolismo , Activación TranscripcionalRESUMEN
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. When compared at the same temperature the enthalpic and entropic contributions the CG base pair are less than that of the AT pair - not more as previously assumed from the extra hydrogen bond. Thus the stabilizing effect of the CG base pair comes from its smaller entropic contribution. The greater enthalpic and entropic contributions of the AT pair result from water fixed by its polar groups in the minor groove of DNA. This water is also responsible for the so-called "nearest-neighbour effects" used to explain the sequence-dependent stabilities of DNA duplexes. Removal of this water by binding DBDs to the minor groove makes this an entropy driven process, in contrast to major groove binding which is enthalpy driven. Analysis of the forces involved in maintaining DNA-DBD complexes shows that specificity of DBD binding is provided by enthalpic interactions, while the electrostatic component that results from counter-ion dispersal is entirely entropic and not sequence-specific. Although the DNA double helix is a rather rigid construction, binding of DBDs to its minor groove often results in considerable DNA bending without the expenditure of significant free energy. This suggests that the rigidity of the DNA duplex comes largely from the water fixed to AT pairs in the minor groove, the loss of which then enables sharp bending.