Structure of the Potassium Form of CGCGAATTCGCG: DNA Deformation by Electrostatic Collapse around Inorganic Cations ,

177

138

The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (e r ∼ 2-4), we found that the DNA dielectric constant is ∼8, considerably higher than the value of ∼3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson-Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques.DNA-ligand binding | DNA packaging | atomic force microscopy | atomistic simulations | Poisson-Boltzmann equation E lectrostatic forces have long been recognized to inherently influence the DNA structure and interactions (1, 2) including DNA bending and folding (3, 4), DNA packaging (5-7), and DNA-ligand recognition (8-10), owing to the high charge density of the DNA molecule. In particular, the crucial role of coulombic forces in the binding affinity of molecules to a specific DNA sequence, such as clinically important drugs into minor grooves (9) and protein complexes into major grooves (11), has been well established (12). However, although detailed knowledge of DNA-DNA and DNA-ligand interactions can nowadays be obtained by high-resolution structural techniques, energetic analysis remains experimentally challenging because it requires quantification of the different contributions to such interactions, in particular the electrostatic energy term.To study the DNA electrostatics, theoretical methods are normally used. All of them require to precisely know the DNA polarization properties in addition to the detailed molecular structure and charge distribution (13-15). In the commonly used mean-field approximation, such as the Poisson-Boltzmann theory, this means including explicitly or implicitly the dielectric constant of DNA, « DNA , a measure of the screening of coulombic forces between charges due to the presence of the DNA molecule. However, despite its crucial impact, the dielectric constant of DNA has remained unknown so far. In the absence of a precise knowledge of « DNA , theoretical models typically assume DNA to be a low-polarizable medium with « DNA ∼ 2-4 like dry proteins surrounded by a highly screening solvent with the dielectric constant of the bulk water ∼80 (13-17). However, this is a simplified picture that lacks experimental validation and introduces a major source of uncertainty, which can give completely biased results in theoretical met...

Section: Resultsmentioning

confidence: 67%

Direct measurement of the dielectric polarization properties of DNA

Cuervo

Dans

Carrascosa

et al. 2014

177

138

“…The crystallographic evidence in favor of this proposition is based entirely on cryogenic diffraction data (36)(37)(38). 23 Na and 87 Rb magnetic relaxation dispersion studies (39,40) of DNA in solution at ambient temperatures indicate that ion binding is weak (partial occupancy) and relatively short-lived (submicrosecond at 277 K), so the thermodynamic and kinetic parameters should fall in the range of values examined here.…”

Section: Discussionmentioning

confidence: 86%

Biomolecular cryocrystallography: Structural changes during flash-cooling

Halle

2004

194

143

To minimize radiation damage, crystal structures of biological macromolecules are usually determined after rapid cooling to cryogenic temperatures, some 150 -200 K below the normal physiological range. The biological relevance of such structures relies on the assumption that flash-cooling is sufficiently fast to kinetically trap the macromolecule and associated solvent in a room-temperature equilibrium state. To test this assumption, we use a two-state model to calculate the structural changes expected during rapid cooling of a typical protein crystal. The analysis indicates that many degrees of freedom in a flash-cooled protein crystal are quenched at temperatures near 200 K, where local conformational and association equilibria may be strongly shifted toward low-enthalpy states. Such cryoartifacts should be most important for strongly solvent-coupled processes, such as hydration of nonpolar cavities and surface regions, conformational switching of solventexposed side chains, and weak ligand binding. The dynamic quenching that emerges from the model considered here can also rationalize the glass transition associated with the atomic fluctuations in the protein.protein structure ͉ protein hydration ͉ protein glass transition ͉ x-ray diffraction

“…R-free was calculated with 5% of the data excluded from the refinement. of different cations (Na ϩ , K ϩ , Mg 2ϩ , Ca 2ϩ ) were determined at high resolution (29)(30)(31)(32)(33)(34)(35), and several of them were isomorphous to the Dickerson-Drew dodecamer (36). The superpositioning of such structures with the present one is shown in Fig.…”

Section: Resultsmentioning

confidence: 92%

DNA bending by an adenine–thymine tract and its role in gene regulation

Hizver

Rozenberg

Frolow

et al. 2001

140

153

To gain insight into the structural basis of DNA bending by adenine-thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-Å resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA bending by such sequence motifs. A comparative analysis of E2-DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein-DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.A-tract ͉ papillomavirus E2-DNA target ͉ transcriptional control T he relationship between DNA sequence, structure, and function has been studied and discussed extensively for the last 20 years. A particular effort has been directed toward the structural elucidation of short runs of four to six adeninethymine residues, known as A-tracts, in an attempt to reveal the structural basis of DNA curvature induced by such sequence motifs when they are inserted in phase with the helical periodicity (1, 2). Despite numerous efforts, including x-ray crystallographic and solution studies, the structural basis of A-tractinduced bending has remained enigmatic (3). Because no single structure could explain the whole phenomenon, it was necessary to rely on models, several of which had been proposed (4). They generally conform to the gel migration data, which suggest that the center of curvature is toward the minor groove of the A-tracts and toward the major groove of the intervening general sequences (5). However, they differ substantially in the details of the stereochemical origin of curvature. This issue is of particular biological significance, as sequence-dependent DNA curvature or bending is an important determinant of DNA recognition by proteins (6).The E2 regulatory system of human papillomaviruses provides an example where sequence-specific binding of proteins to A-tracts is crucial to the organism's life cycle. The E2 proteins from all viral strains activate or repress transcription in a context-dependent manner and are required for the initiation of replication in vivo. Their function depends on sequence-specific binding to a highly conserved 12-bp sequence of the general form ACCGNNNNCGGT, where N4 is variable (7,8). However, the E2 binding sites in the human papillomavirus (HPV) genomes, including the cancer-related strains HPV-16 and HPV-18 (9), exhibit a further level of specificity in the interaction with their cognate E2 proteins. This specificity is manifested by th...