Helix-coil transition theory including long-range electrostatic interactions: application to globular proteins

An extension of the Zimm–Bragg two-state theory for the helix–coil transition in polypeptides, which takes into account the effect of peptide charge–dipole interactions on helix stability, is presented. This new theory incorporates these interactions in an expression that is parameterized on recently obtained experimental data on polypeptides for which electrostatic effects are known to influence helix content. Unlike previous two-state or multistate models, which are parameterized on protein x-ray data, the present theoretical treatment in independent of such protein data. The theoretical model is applied to a series of peptides derived from the C-peptide of ribonuclease A, which have been the object of recent spectroscopic studies. The new theoretical approach can account for most of the structural information derived from studies of these C-peptides, and for overall average helix probabilities that are close in magnitude to those observed for these polypeptides in solution. An application of this new formulation for the prediction of the locations of α-helices in globular proteins from their amino acid sequence is also presented.     

Powering DNA repair through substrate electrostatic interactions

The reaction catalyzed by the DNA repair enzyme uracil DNA glycosylase (UDG) proceeds through an unprecedented stepwise mechanism involving a positively charged oxacarbenium ion sugar and uracil anion leaving group. Here we use a novel approach to evaluate the catalytic contribution of electrostatic interactions between four essential phosphodiester groups of the DNA substrate and the cationic transition state. Our strategy was to substitute each of these phosphate groups with an uncharged (R)- or (S)-methylphosphonate linkage (MeP). We then compared the damaging effects of these methylphosphonate substitutions on catalysis with their damaging effects on binding of a cationic 1-azadeoxyribose (1-aza-dR(+)) oxacarbenium ion analogue to the UDG-uracil anion binary complex. A plot of log k(cat)/K(m) for the series of MeP-substituted substrates against log K(D) for binding of the 1-aza-dR(+) inhibitors gives a linear correlation of unit slope, confirming that the electronic features of the transition state resemble that of the 1-aza-dR(+), and that the anionic backbone of DNA is used in transition state stabilization. We estimate that all of the combined phosphodiester interactions with the substrate contribute 6-8 kcal/mol toward lowering the activation barrier, a stabilization that is significant compared to the 16 kcal/mol catalytic power of UDG. However, unlike groups of the enzyme that selectively stabilize the charged transition state by an estimated 7 kcal/mol, these phosphodiester groups also interact strongly in the ground state. To our knowledge, these results provide the first experimental evidence for electrostatic stabilization of a charged enzymatic transition state and intermediate using the anionic backbone of DNA.     

The electrostatic contribution to DNA base-stacking interactions.

Base-stacking and phosphate-phosphate interactions in B-DNA are studied using the finite difference Poisson-Boltzmann equation. Interaction energies and dielectric constants are calculated and compared to the predictions of simple dielectric models. No extant simple dielectric model adequately describes phosphate-phosphate interactions. Electrostatic effects contribute negligibly to the sequence and conformational dependence of base-stacking interactions. Electrostatic base-stacking interactions can be adequately modeled using the Hingerty screening function. The repulsive and dispersive Lennard-Jones interactions dominate the dependence of the stacking interactions on roll, tilt, twist, and propellor. The Lennard-Jones stacking energy in ideal B-DNA is found to be essentially independent of sequence.     

On the truncation of long-range electrostatic interactions in DNA

Long-range interactions are known to play an important role in highly polar biomolecules like DNA. In molecular dynamics simulations of nucleic acids and proteins, an accurate treatment of the long-range interactions are crucial for achieving stable nanosecond trajectories. In this report, we evaluate the structural and dynamic effects on a highly charged oligonucleotide in aqueous solution from different long-range truncation methods. Two group-based truncation methods, one with a switching function and one with a force-switching function were found to fail to give accurate stable trajectories close to the crystal structure. For these group-based truncation methods, large root mean square (rms) deviations from the initial structure were obtained and severe distortions of the oligonucleotide were observed. Another group-based truncation scheme, which used an abrupt truncation at 8. 0 A or at 12.0 A was also investigated. For the short cutoff distance, the conformations deviated far away from the initial structure and were significantly distorted. However, for the longer cutoff, where all necessary electrostatic interactions were included, the trajectory was quite stable. For the particle mesh Ewald (PME) truncation method, a stable DNA simulation with a heavy atom rms deviation of 1.5 A was obtained. The atom-based truncation methods also resulted in stable trajectories, according to the rms deviation from the initial B-DNA structure, of between 1.5 and 1.7 A for the heavy atoms. In these stable simulations, the heavy atom rms deviations were approximately 0.6-1.0 A lower for the bases than for the backbone. An increase of the cutoff radius from 8 to 12 A decreased the rms deviation by approximately 0.2 A for the atom-based truncation method with a force-shifting function, but increased the computational time by a factor of 2. Increasing the cutoff from 12 to 18 A for the atom-based truncation method with a force-shifting function requires 2-3 times more computational time, but did not significantly change the rms deviation. Similar rms deviations from the initial structure were found for the atom-based method with a force-shifting function and for the PME method. The computational cost was longer for the PME method with a cutoff of 12. 0 A for the direct space nonbonded calculations than for the atom-based truncation method with a force-shifting function and a cutoff of 12.0 A. If a nonperiodic boundary, e.g., a spherical boundary, was used, a considerable speedup could be achieved. From the rms fluctuations, the terminal nucleotides and especially the cytidines were found to be more flexible than the nonterminal nucleotides. The B-DNA form of the oligonucleotide was maintained throughout the simulations and is judged to depend on the parameters of the energy function and not on the truncation method used to handle the long-range electrostatic interactions. To perform accurate and stable simulations of highly charged biological macromolecules, we recommend that the atom-based force-shift method or the PME method should be used for the long-range electrostatics interactions.     

Free energy calculation on base specificity of drug--DNA interactions: application to daunomycin and acridine intercalation into DNA

We present the results of free energy perturbation/molecular dynamics studies on B-DNA.daunomycin and B-DNA.9-aminoacridine complexes as well as on B-DNA itself in order to calculate the free energy differences between complexes having different base pair sequences. The results generally reproduce the trends observed experimentally, i.e., preferences of acridine and daunomycin to bind to a specific base sequence in the DNA. This is encouraging, given the simplicity of the molecular mechanical/dynamical model in which solvent is not explicitly included.     

DNA A-tracts bending: polarization effects on electrostatic interactions across their minor groove

Bending by the DNA A-tracts constitutes a contentious issue, suggesting deficiencies in the physics employed so far. Here, we inquire as to the importance in this bending of many-body polarization effects on the electrostatic interactions across their narrow minor groove. We have done this on the basis of the findings of Jarque and Buckingham who developed a procedure based on a Monte Carlo simulation for two charges of the same sign embedded in a polarizable medium. Remarkably, the present analysis reveals that for compact DNA conformations, which result from dynamic effects, an overall attractive interaction operates between the phosphate charges; this interaction is especially strong for the narrow minor groove of the A-tracts, suggesting a tendency for DNA to bend toward this groove. This tendency is in agreement with the conclusions of electrophoretic and NMR solution studies. The present analysis is also consistent with the experimental observations that the minor groove is much more easily compressible than the major groove and the bending propensity of the A-tracts is greatly reduced at “premelting” temperatures. By contrast, the dielectric screening model predicts a repulsion between the phosphate charges and is not consistent with the aforementioned bending tendency or experimental observations.     

Base-stacking interactions in double-helical DNA structures: experiment versus theory

Atom-atom potential energy calculations have been undertaken for deriving stacking energies in double-helical structures. A comparison between the energy patterns of A- and B-type double-helical fragments determined by single-crystal X-ray diffraction methods versus idealized uniform models based on fibre diffraction data shows that the van der Waals stacking energy is largely sensitive to local changes in the relative orientation of adjacent base pairs. The sequence-dependent conformational variability observed in the high-resolution structures appears to be a consequence of the equipartitioning of the stacking energy along the double helix. The large energy variations expected for a uniform structure are dampened considerably in the observed structures by means of local changes in conformational features such as helix rotation and roll angles between base pairs.     

Stretching single-stranded DNA: interplay of electrostatic, base-pairing, and base-pair stacking interactions.

Recent single-macromolecule observations revealed that the force/extension characteristics of single-stranded DNA (ssDNA) are closely related to solution ionic concentration and DNA sequence composition. To understand this, we studied the elastic property of ssDNA through the Monte Carlo implementation of a modified freely jointed chain (FJC), with electrostatic, base-pairing, and base-pair stacking interactions all incorporated. The simulated force-extension profiles for both random and designed sequences have attained quantitative agreements with the experimental data. In low-salt solution, electrostatic interaction dominates, and at low forces, the molecule can be more easily aligned than an unmodified FJC. In high-salt solution, secondary hairpin structure appears in ssDNA by the formation of base pairs between complementary bases, and external stretching causes a hairpin-coil structural transition, which is continuous for ssDNA made of random sequences. In designed sequences such as poly(dA-dT) and poly(dG-dC), the stacking potential between base pairs encourages the aggregation of base pairs into bulk hairpins and makes the hairpin-coil transition a discontinuous (first-order) process. The sensitivity of elongation to the base-pairing rule is also investigated. The comparison of modeling calculations and the experimental data suggests that the base pairing of single-stranded polynucleotide molecules tends to form a nested and independent planar hairpin structure rather than a random intersecting pattern.     

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.     

Chemically modified DNA substrates implicate the importance of electrostatic interactions for DNA unwinding by Dda helicase

Helicase-catalyzed disruption of double-stranded nucleic acid is vital to DNA replication, recombination, and repair in all forms of life. The relative influence of specific chemical interactions between helicase and the substrate over a series of multistep catalytic events is still being defined. To this end, three modified DNA oligonucleotides were designed to serve as substrates for the bacteriophage T4 helicase, Dda. A 5'-DNA-PNA-DNA-3' chimera was synthesized, thereby, conferring both a loss of charge and altering the conformational flexibility of the oligonucleotide. The second modified oligonucleotide possessed a single methylphosphonate replacement on the phosphate backbone, creating a gap in the charge distribution of the substrate. The third modification introduced an abasic site into the oligonucleotide sequence. This abasic site retains the charge distribution of the normal DNA substrate yet alters the conformational flexibility of the oligonucleotide. The loss of a base also serves to disrupt the hydrogen-bonding lattice, the intramolecular base-stacking interactions, as well as the intermolecular base-stacking interactions between aromatic amino acid side chains and the substrate. Our results indicate that a gap in the charge distribution along the backbone of the substrate has a more pronounced effect upon helicase-catalyzed unwinding than does the loss of a single base. While all three substrates exhibited some degree of inhibition, analysis of both pre-steady-state and excess enzyme experiments places a greater value upon the electrostatic interactions between helicase and the substrate.     

Variable electrostatic interaction between DNA and coat protein in filamentous bacteriophage assembly

A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the DNA of the class I filamentous bacteriophage fd, which infects Escherichia coli. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter, generating plasmid pKf8P. Bacteriophage fd gene VIII was similarly cloned into the plasmid pEMBL9+, enabling it to be subjected to site-directed mutagenesis. By this means the positively charged lysine residue at position 48, one of four positively charged residues near the C terminus of the protein, was turned into a negatively charged glutamic acid residue. The mutated fd gene VIII was cloned back from the pEMBL plasmid into the expression plasmid pKK223-3, creating plasmid pKE48. In the presence of the inducer isopropyl-beta-D-thiogalactoside, the wild-type and mutated coat protein genes were strongly expressed in E. coli TG1 cells transformed with plasmids pKf8P and pKE48, respectively, and the product procoat proteins underwent processing and insertion into the E. coli cell inner membrane. A net positive charge of only 2 on the side-chains in the C-terminal region is evidently sufficient for this initial stage of the virus assembly process. However, the mutated coat protein could not encapsidate the DNA of bacteriophage R252, an fd bacteriophage carrying an amber mutation in its own gene VIII, when tested on non-suppressor strains of E. coli. On the other hand, elongated hybrid bacteriophage particles could be generated whose capsids contained mixtures of wild-type (K48) and mutant (E48) subunits. This suggests that the defect in assembly may occur at the initiation rather than the elongation step(s) in virus assembly. Other mutations of lysine-48 that removed or reversed the positive charge at this position in the C-terminal region of the coat protein were also found to lead to the production of commensurately longer bacteriophage particles. Taken together, these results indicate direct electrostatic interaction between the DNA and the coat protein in the capsid and support a model of non-specific binding between DNA and coat protein subunits with a stoicheiometry that can be varied during assembly.     

Influence of electrostatic interactions on the sugar–phosphate backbone conformation in DNA

The influence of sugar ring flexibility in DNA on the mechanism of the B ? A conformational transition is studied. The dipole moment of the deoxyribose as a function of its puckered states is calculated by the quantum-mechanical method using the MINDO/3 approximation. The interaction of the sugar dipole with the neighbor molecular groups in polynucleotide chains is estimated. The sugar dipole interaction with phosphate groups and counterions is shown to be strong and capable to deform the pseudorotation potential of deoxyribose. The effective pseudorotation potential of sugar ring in the B and A helices is obtained. The results are used to explain the behavior of Raman bands in the region of sugar–phosphate vibrations. The mechanism of the effect of electrostatic forces on the sugar–phosphate backbone conformation, which is essential for the B ? A and other structure transitions, is offered. © 1993 John Wiley & Sons, Inc.     

2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: metal-ion and base sequence effects.

Metal-ion and sequence dependent changes in the stacking interactions of bases surrounding abasic (AB) sites in 10 different DNA duplexes were examined by incorporating the fluorescent nucleotide probe 2-aminopurine (2-AP), opposite to the site (AB-APopp) or adjacent to the site (AB-APadj) on either strand. A detailed study of the fluorescence emission and excitation spectra of these AB duplexes and their corresponding parent duplexes indicates that AB-APoppis significantly less stacked than 2-AP in the corresponding normal duplex. In general, AB-APadjon the AB strand is stacked, but AB-APadjon the opposite strand shows destabilized stacking interactions. The results also indicate that divalent cation binding to the AB duplexes contributes to destabilizaton of the base stacking interactions of AB-APopp, but has little or no effect on the stacking interactions of AB-APadj. Consistent with these results, the fluorescence of AB-APoppis 18-30-fold more sensitive to an externally added quenching agent than the parent normal duplex. When uracil DNA glycosylase binds to AB-APoppin the presence of 2.5 mM MgCl2, a 3-fold decrease in fluorescence is observed ( K d = 400 +/- 90 nM) indicating that the unstacked 2-APoppbecomes more stacked upon binding. On the basis of these fluorescence studies a model for the local base stacking interactions at these AB sites is proposed.     

Phenomenological theory of GC/AT pressure on DNA base composition

Summary We present a phenomenological theory expressing the constraints operating on the (G+C) contents of the three codon positions, i.e., first, second, and third bases of codons, by using the smallest number of constraint parameters having clear physical and genetic meaning. Theoretical curves displaying base composition at each of the three codon sites are given. The agreement between the theoretical curves and the data points of 1277 genes is quite good irrespective of the species from which the DNAs originated; the curves might be universal ones and the constraint parameters might have general biological meanings in relation to the DNA/RNA and protein functions.     

Complementary recognition in condensed DNA: accelerated DNA renaturation.

The functional consequences of DNA condensation are investigated. The recognition of complementary strands is profoundly modified by this critical phenomenon. (1) Condensation of denatured DNA greatly accelerates the kinetics of DNA renaturation. We propose a unifying explanation for the effects of several accelerating solvents studied here including polymers, di- and multivalent cations, as well as effects seen with the phenol emulsions and single-stranded nucleic acid binding proteins. Optimal conditions for renaturation at or above the calculated three dimensional diffusion limit are theoretically consistent with a limited search space in the condensed phases. (2) In addition to these effects on association of two single strands, similar condensation acceleration effects can be seen in strand exchange experiments with double stranded DNA without proteins. These may model a mechanism of recombinational protein function.     

Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA

Bacteriophage fd gene VIII, which encodes the major capsid protein, was mutated to convert the serine residue at position 47 to a lysine residue (S47K), thereby increasing the number of positively charged residues in the C-terminal region of the protein from four to five. The S47K coat protein underwent correct membrane insertion and processing but could not encapsidate the viral DNA, nor was it incorporated detectably with wild-type coat proteins into hybrid bacteriophage particles. However, hybrid virions could be constructed from the S47K coat protein and a second mutant coat protein, K48Q, the latter containing only three lysine residues in its C-terminal region. K48Q phage particles are approximately 35% longer than wild-type. Introducing the S47K protein shortened these particles, the S47K/K48Q hybrids exhibiting a range of lengths between those of K48Q and wild-type. These results indicate that filamentous bacteriophage length (and the DNA packaging underlying it) are regulated by unusually flexible electrostatic interactions between the C-terminal domain of the coat protein and the DNA. They strongly suggest that wild-type bacteriophage fd makes optimal use of the minimum number of coat protein subunits to package the DNA compactly.     

The effect of electrostatic interactions on the conformation of the sugar-phosphate backbone in DNA]

The influence of sugar ring flexibility in DNA on the mechanism of the B<-->A conformational transition is studied. The dipole moment of the deoxyribose as a function of its puckered states is calculated by the quantum-mechanical method using the MINDO/3 approximation. The interaction of the sugar dipole with the neighbour molecular groups in polynucleotide chain is estimated. The sugar dipole interaction witch phosphate groups and counterions is shown to be strong and capable to deform the pseudorotation potential of deoxyribose. The effective pseudorotation potential of sugar ring in the B- and A-helices is obtained. The results are used to explain the behaviour of Raman bands in the region of sugar-phosphate vibrations. The mechanism of the effect of electrostatic forces on the sugar-phosphate backbone conformation which is essential for the B<-->A and other structure transitions is offered.