The effects of thioketo substitution upon uracil-adenine interactions in polyribonucleotides. Synthesis and properties of the alternating polynucleotides poly(r(A-s2U)) and poly(r(A-s2s4U)).

The polynucleotides poly[r(A-s-2U)] and poly]r(A-s2s4U)] have been synthesized and characterized by nearest-neighbour analysis, sedimentation analysis as well as spectroscopic techniques. Absorption-temperature profile and absorption-pH profile of poly[r(A-s-2U)] did not reveal a structural transition between 10 and 95 degrees C even at low ionic strength, although a variety of properties indicated a helical structure of poly[r(A-s-2U)]: remarkable hyperchromicity of the absorption spectrum, circular dichroic spectrum displaying extrema of large amplitudes, resistance against hydrolysis by ribonuclease and interaction with ethidium bromide in a manner which is characteristic of helical polynucleotides. Our results show that interactions of the type A-s-2U and A-s-2s-4U do in fact exist in helical polynucleotides. The properties of poly]r(As-2U)] furthermore demonstrate the general stabilizing effect of 2-thioketopyrimidine bases in helical polynucleotides by virtue of vertical stacking interactions with neighbouring pyrimiding and purine bases.     

Na+ effects on transition of DNA and polynucleotides of variable linear charge density.

A range of linear charge densities of the ordered and disordered forms of DNA or polynucleotides can be obtained experimentally by acid or alkaline titration, or by the investigation of unusual complexes involving protonated bases or three-stranded helices. The variation of melting temperatures with Na⁺ concentration for various of these systems is known and in some cases is complemented by structural and thermodynamic information. We have extended the condensation–screening theory of Manning [Biopolymers, 11 , 937–955 (1972)] to these systems. The stabilizing and destabilizing effects of Na⁺ (condensation and screening, respectively) and be independently varied, and the theory is successful in predicting the qualitative (in some cases, quanittative) behaviour that is observed. Comparison of theory and experiment indicates that the axial phosphate distance b for single-stranded polynucleotides increases with increasing pH. Values of the critical parameter ξ are obtained for the various polynucleotide structures. These values are essential for an understanding of ionic effects on charged ligand–polynucleotide interactions.     

Formation of ternary complexes involving zinc or copper ions, polynucleotides, and polypeptides containing glutamic acid and tyrosine residues

Metal ions such as Zn²⁺ and Cu²⁺ can mediate interactions between copolypeptides (Glu_x, Tyr_y)_n and polynucleotides. CD data show that these ternary complexes are characterized by an unstacking of nucleic acid bases, while the polypeptide adopts an α-helical conformation as observed in the two binary complexes polynucleotide–cation and polypeptide–cation. Fluorescence studies demonstrate that tyrosyl side chains interact with nucleic acid bases in the ternary complexes, leading to a quenching of tyrosine fluorescence.     

Mechanism of RNA-protein interactions in tobacco mosaic virus: analysis of the pH stability of virus protein complexes with synthetic polynucleotides.

TMV-like RNP complexes were reconstituted from TMV protein and synthetic polynucleotides. Analysis of the pH stability of RNP with polynucleotides containing U, G, or their analogues reveals a correlation between the stability of their structure and the pK values of the bases, and indicates that the -NH-CO-groups of U and G are involved in hydrogen bonding with protein. It is suggested that TMV protein has two U- and one G-specific binding sites which, according to the phase position of the protein subunits relative to the origin of TMV assembly (D. Zimmern (1977), Cell 11, 463) are likely to be organized as UGU. The binding of the A and C residues of RNA with TMV protein is nonspecific. TMV protein groups with pK 6.3, 7.5 and 9.7 were found to be essential in the protein-protein interactions in RNP. A group of the protein with pK 8.2 is also involved in RNP stabilization. Both protein-protein interactions and interactions of protein with RNA phosphate groups were shown to be mediated by a conformational change in the protein induced by base binding. The effect of bases on both types of interactions changes in the order G approximately equal to much greater than A, and incorporation of C in RNP proceeds in a compulsory way at the expense of interaction of the neighbouring nucleotide residues in polynucleotides with protein. The data obtained are used to discuss the principles of the cooperativity of the interactions between TMV components and the mechanism of initiation and elongation in TMV self-assembly.     

Circular dichroism of acridine orange bound to DNA

The circular dichroism (CD) spectra of DNA–acridine orange (DNA–AO) complex in the visible region were measured at DNA phosphate-to-dye ratios (P/D) from 1 to 550. The CD spectrum of DNA–AO complex in the P/D ratio between 1 and approximately 40 consists of four components, i.e., positive CD bands centered at 510 and 480 mμ, and negative CD bands at 497 and 468 mμ. The CD bands at 510 and 468 mμ are optimum at P/D = 4, and the change of ε₁ ? ε_r with P/D suggests that both of them are induced from the interaction between dye molecules bound to adjacent DNA binding sites, each of which is composed of four nucleotides. This is supported by the fact that the values of ε₁ ? ε_r for both decrease with increasing temperature or increasing methylene blue concentration added to the complex. The negative Cotton effect at, 497 mμ is most favored at larger P/D ratio (～8), and the suggested assignment is to the interaction between two dye molecules bound with an empty site between them. A positive Cotton effect at 480 mμ is observed at P/D ratio of less than 4 and is optimum at 1. Above P/D ratio of 40, the CD spectrum of the complex can not be resolved into its components and even at sufficiently high P/D ratio (550) the complex shows a small Cotton effect.     

Cooperative interactions in single-strand oligomers of adenylic acid

Optical rotatory dispersion measurements were made on the oligonucleotides (pA)₂, (pA)₄, and (pA)₆ at neutral pH over the temperature range 5–85°C., and compared to similar data for polyriboadenylic acid. The data were interpreted in terms of a temperature-dependent stacking of the bases in the single-strand oligomers, with very little dependence of the degree of stacking on the chain length. These results can be explained by a theory of cooperative stacking. The degrees of freedom available per residue are rotations about the five backbone covalent bonds and the bond connecting the base to the ribose ring. To nucleate a stacking interaction between neighboring bases the backbone sequence must be ordered as must be the two bases. For this stack to grow by one base a backbone sequence must again be ordered, but only one additional base must be ordered. Thus, the degree of freedom of the base with respect to the ribose ring determines the extent of the cooperative effect and hence the effect of chain length. A matrix formulation of the partition function is presented which incorporates this cooperative nature of the interaction and is shown to be in fair agreement with the data. The entropy of ordering a base with respect to the ribose ring is found to be 0.68 e.u., which suggests that the purine has a torsional oscillation when unstacked, but does not have several isoenergetic positions of internal rotation available. The enthalpy of stacking is found to be ?6.5 kcal./mole. A model involving neighbor and next-nearest neighbor interactions could also account for the data. For all practical purposes, the stacking interactions of successive residues can be treated as independent, i.e., the state of one residue is essentially independent of the state of stacking of its neighbors.     

Quantum-mechanical study of the hypochromic effect in polynucleotides. Intra- and interstrand interaction contributions.

The first uv absorption band hypochromism of poly(dA) · poly(dT), poly(dG) · poly(dC), poly(dA), poly(dT), poly(dG), and poly(dC) is calculated with the help of perturbation theory on the basis of monomer characteristics computed by the Pariser-Parr-Pople method taking into account all singly excited configurations. The theoretical results obtained are in good agreement with experimental values of hypochromism. The origin of the hypochromic effect in the double-stranded polynucleotides is investigated. It is shown that intrastrand interactions between the bases make the main contribution to hypochromism (60–76%), while the contribution of the Watson–Crick-pair formation is small (2–12%). The essential part of hypochromism (22–28%) is due to the interstrand interactions between the bases that are not coupled by hydrogen bonds. The discussion of the experimental data shows that the present theoretical investigation could serve as a basis for the correct treatment of experimental data.     

Coprecipitation of thermal lysine-rich proteinoids with polyribonucleotides.

Major variables in interactions between basic thermal proteinoids and homopolyribonucleotides were magnesium concentration in solution (0–40 mM) and mol% lysine in the proteinoid (16–55%). The formation of microparticles was monitored both by the turbidity and by the mass of precipitate formed. Under some conditions, only, was the turbidity reading a reliable indication of the amount of precipitate. Increasing concentration of Mg²⁺ tended to displace proteinoid from the complex with polynucleotide. Of 4 polynucleotides, only polyguanylic acid showed an enhanced precipitation of proteinoid in the presence of Mg²⁺, and then only with those having high lysine contents. At high lysine contents, the amount of proteinoid in the precipitate was inversely proportional to the lysine content of the proteinoids, probably due to decreased sidechain interactions. The precipitation with polynucleotides is partly a function of the amino acid composition of the proteinoid; therefore the interaction of thermal proteinoids with polynucleotides appears to be a tool that can be used to study specificities of interactions between proteins and nucleic acids.     

Optically detected magnetic resonance of tryptophan residues in Escherichia coli ssb gene product and E. coli plasmid-encoded single-stranded DNA-binding proteins and their complexes with poly(deoxythymidylic) acid

Optically detected magnetic resonance (ODMR) spectroscopy has been applied to several single-stranded DNA-binding (SSB) proteins encoded by conjugative plasmids of enteric bacteria. Fluorimetric equilibrium binding isotherms confirm their preferential binding to single-stranded DNA and polynucleotides and reveal a limited protein solubility at low ionic strength. The plasmid SSB-like proteins show the highest affinity for polydeoxythymidylic acid; these complexes are the least sensitive to disruption by salt. ODMR data on these complexes suggest the existence of stacking interactions between tryptophan residue(s) and thymine bases, as evidenced by spectral red shifts of the tryptophan phosphorescence 0,0 band, reduction of the magnitude of D zero field splitting parameter, and a dramatic reversal of the polarity of the ODMR signals. Wavelength-selected ODMR results point to the existence of two distinct tryptophan sites in these complexes. The triplet state properties of the red-shifted site are drastically altered by its interaction with the thymine bases. The chromosomal Escherichia coli SSB protein-poly(dT) complex shows an additional tryptophan site with zero field splitting parameters similar to those of the free protein. This site can be attributed to Trp-135, which is missing in each of the other plasmid SSB proteins, suggesting that this particular residue is not involved in the interaction with polynucleotides.     

Binding site identification of anticancer drug gefitinib to HSA and DNA in the presence of five different probes

This study was carried out to evaluate the binding interaction of gefitinib (GEF) with human serum albumin (HSA) and calf thymus DNA (ct-DNA) using fluorescence, UV–Visible, zeta potential measurements and molecular docking methods in order to understand its pharmacokinetic mechanism. By increasing the temperature, a steady decrease in Stern–Volmer quenching constants was observed for HSA binding properties; this indicates a static type of fluorescence quenching. Negative values were calculated for Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) changes, indicating that the reaction is spontaneous and enthalpy-driven. Probe competitive experimental results showed that GEF contains the same binding site as warfarin and are consistent with modeling results. The zeta potential of the HSA increased with increasing GEF, which represents the presence of electrostatic interactions in the system. DNA binding properties were investigated in the presence of three probes. The experimental results showed that by increasing GEF to DNA-AO (acridine-orange) and DNA-MB (methylene-blue) system, the fluorescence intensity and absorbance spectra had no considerable change. Furthermore, with the addition of GEF to DNA, the zeta potential decreased gradually, indicating that the hydrophobic interaction between the GEF and the bases of DNA is the major factor. Thus, GEF can bind to DNA via a groove binding mode. It was also found that GEF entered into the minor groove in the A–T rich region of DNA fragment and bind via van der-Waals forces and three H-bond with double strands of DNA. This is in good agreement with experimental results.     

Stacking interactions of tryptophan residues and nucleotide bases in complexes formed between Escherichia coli single-stranded DNA binding protein and heavy atom-modified poly(uridylic) acid. A study by optically detected magnetic resonance spectroscopy

The complexes formed between Escherichia coli single-stranded DNA binding protein (SSBP) and the heavy atom-modified single-stranded polynucleotides poly(5-BrU) and poly(5-HgU) are investigated using optically detected magnetic resonance (ODMR) methods. In these complexes the triplet state properties of the tryptophan residues are subjected to the external heavy atom effect generated by bromine and mercury atoms and are characterized by a shortened triplet state lifetime and the appearance of the otherwise dark [D] + [E] slow passage ODMR signal. These features provide direct evidence for close range interactions between tryptophan residue(s) and the nucleotide bases in the complexes. The extent of the triplet state lifetime reduction in the case of the SSBP-poly(5-HgU) complex together with steric considerations of the complex structure is consistent only with a van der Waals contact between the perturbed molecule and the heavy atom perturber by means of a stacking interaction. Fast passage ODMR measurements show a lifetime for a sublevel of the perturbed tryptophan chromophore(s) in this complex on the order of 1 ms. The amplitude-modulated phosphorescence microwave double resonance technique captures selectively the broadened and red-shifted phosphorescence spectrum of the heavy atom-perturbed tryptophan residue(s). This work supports a model for the binding of SSBP to single-stranded polynucleotides in which the bases are inserted into hydrophobic regions of the protein, where they are likely to undergo stacking interactions with the indole moiety of buried tryptophan residues.     

Warming up the system: higher predator feeding rates but lower energetic efficiencies

Predictions on the consequences of the rapidly increasing atmospheric CO₂ levels and associated climate warming for population dynamics, ecological community structure and ecosystem functioning depend on mechanistic energetic models of temperature effects on populations and their interactions. However, such mechanistic approaches combining warming effects on metabolic (energy loss of organisms) and feeding rates (energy gain by organisms) remain a key, yet elusive, goal. Aiming to fill this void, we studied the metabolic rates and functional responses of three differently sized, predatory ground beetles on one mobile and one more resident prey species across a temperature gradient (5, 10, 15, 20, 25 and 30 °C). Synthesizing metabolic and functional‐response theory, we develop novel mechanistic predictions how predator–prey interaction strengths (i.e., functional responses) should respond to warming. Corroborating prior theory, warming caused strong increases in metabolism and decreases in handling time. Consistent with our novel model, we found increases in predator attack rates on a mobile prey, whereas attack rates on a mostly resident prey remained constant across the temperature gradient. Together, these results provide critically important information that environmental warming generally increases the direct short‐term per capita interaction strengths between predators and their prey as described by functional‐response models. Nevertheless, the several fold stronger increase in metabolism with warming caused decreases in energetic efficiencies (ratio of per capita feeding rate to metabolic rate) for all predator–prey interactions. This implies that warming of natural ecosystems may dampen predator–prey oscillations thus stabilizing their dynamics. The severe long‐term implications; however, include predator starvation due to energetic inefficiency despite abundant resources.     

Warming affects different components of plant–herbivore interaction in a simplified community but not net interaction strength

Global warming impacts natural communities through effects on performance of individual species and through changes in the strength of interactions between them. While there is a body of evidence of the former, we lack experimental evidence on potential changes in interaction strengths. Knowledge about multispecies interactions is fundamental to understand the regulation of biodiversity and the impact of climate change on communities. This study investigated the effect of warming on a simplified community consisting of three species: rosy apple aphid Dysaphis plantaginea feeding on plantain, Plantago lanceolata, and a heterospecific neighbouring plant species, perennial ryegrass, Lolium perenne. The aphid does not feed on L. perenne. The experimental design consisted of monocultures and mixtures of L. perenne and P. lanceolata at three temperature levels. We did not find indication for indirect temperature effects on D. plantaginea through changes in leaf nitrogen or relative water content. However, experimental warming affected the life history traits of the aphid directly, in a non‐linear manner. Aphids performed best at moderate warming, where they grew faster and had a shorter generation time. In spite of the increased population growth of the aphids under warming, the herbivory rates were not changed and consequently the plant–herbivore interaction was not altered under warming. This suggests reduced consumption rates at higher temperature. Also plant competition affected the aphids but through an interaction with temperature. We provide proof‐of‐concept that net interactions between plants and herbivores should not change under warming despite direct effects of warming on herbivores when plant–plant interaction are considered. Our study stresses the importance of indirect non–trophic interactions as an additional layer of complexity to improve our understanding of how trophic interactions will alter under climate change.     

Theoretical study of the hypochromic effect in polynucleotides]

The first UV-absorption band hypochromicity of poly(dA)-poly(dT), poly(dG)-poly(dC), poly(dA), poly(dT), poly(dG), poly(dC), is calculated with the help of the perturbation theory. The wave functions of the bases are computed by Pariser--Parr--Pople's method taking into account all the singly excited configurations. The results obtained show a good correlation between the theoretical and experimental values of hypochromicity. A considerable influence of the vaccum electron transitions on the hypochromicity of polynucleotides is revealed. The origin of the hypochromic effect in the double-stranded polynucleotides is investigated. It is shown that intrastrand interactions between the bases make the main contribution to hypochromicity (60-76%), while the contribution of the Watson-Crick pairs is small (2-12%). The essential part of hypochromicity (22-28%) is due to the interstrand interactions between the bases which are not coupled by hydrogen bonds. The discussion of the experimental data shows that the present theoretical investigation could serve as a basis for correct treatment of experimental results.     

Calorimetric determination of the heat capacity changes associated with the conformational transitions of polyriboadenylic acid and polyribouridylic acid

The heat capacities of the single-stranded and double-stranded forms of polyadenylic acid, polyuridylic acid, and poly(uridylic and adenylic acid) were determined with a drop heat capacity calorimeter. In addition, the temperature dependence of the apparent partial heat capacity (?Cp) was measured with a newly developed differential scanning calorimeter. The calculated ΔCp at 28°C for the transition poly(A)·poly(A) ? 2 poly(A) was found to be 165 ± 24 cal/Kmol-base pair, compared with a value of 140 ± 28 for the transition poly(A)·poly(U) ? poly(A) + poly(U). The temperature dependence of ?Cp of single-stranded poly(U) was consistent with the conclusion that it is totally unstacked at temperatures above 15°C. The temperature dependence of ?Cp of single-stranded poly(A) was used to determine the base-stacking parameters for poly(A). The experimental results are consistent with a stacking enthalpy change of ?8.5 ± 0.1 kcal/mol bases and a cooperativity parameter σ of 0.57 ± 0.03 for the stacking of adenine bases. These results demonstrate that the heat capacity of single-stranded polynucleotides is greater than that of the double-stranded forms. This increased heat capacity is mainly the result of the temperature dependence of the base-stacking interactions in the single-stranded form.     

Fluorescence and binding properties of phenazine derivatives in complexes with polynucleotides of various base compositions and secondary structures

The interactions of two phenazine derivatives, one with a neutral chromophore (glycoside) and the other with a cationic one (quaternary salt), with various synthetic single- and double-stranded polynucleotides and natural DNA were studied by fluorescence techniques, conducting measurements of steady-state fluorescence intensity and polarization degree as well as fluorescence lifetime. These dyes show fluorescence quenching upon intercalation into the GC sequences of the double-stranded nucleic acids and an increase in fluorescence emission and lifetime upon incorporation into the AT and AU sequences. GC base pairs in continuous deoxynucleotide sequences were found to be preferred as binding sites for both phenazines, in contrast to AT base pairs. On the contrary, the continuous ribonucleotide GC sequence binds the phenazines more weakly than does the AU sequence. With regard to the interaction of the phenazines with single-stranded polynucleotides, a stacking interaction of the dye chromophores with the nucleic bases was observed. In that case the guanine residue quenches the cationic phenazine fluorescence, while the stacking interaction with the other bases results in an increase in the fluorescence quantum yield. Unlike the cationic dye, the fluorescence of the neutral phenazine was quenched by both purine bases.     

A specific model for the conformation of single-stranded polynucleotides in complex with the helix-destabilizing protein GP32 of bacteriophage T4

The CD and absorption (OD) spectra of single-stranded nucleic acids in complex with the helix-destabilizing protein of either bacteriophage T4 (GP32) or bacteriophage fd (GP5) show similar and unusual features for all polynucleotides investigated. The change in the CD spectra between 310 and 240 nm is in all cases characterized by a considerable decrease in the positive band, while the negative band (if present) remains relatively intense. These changes are different from those due to temperature or solvent denaturation and, moreover, cannot be induced by the binding of simple oligopeptides. Absorption measurements show that all polynucleotides remain hypochromic in the complex. Both CD and OD spectra point to a specific and probably similar conformation in complex for all polynucleotides with substantial interactions between the bases. The spectral properties are almost temperature independent (0–40°C). Therefore, we conclude that the conformation must be regular and rigid. To investigate the relation between these optical properties and the specific polynucleotide structure, CD and OD spectra were calculated for an adenine hexamer over a wide range of the conformational parameters. It appears that the calculated CD intensity is not very sensitive to an increase in the axial increment and that many different conformations can give rise to more or less similar CD spectra. However, simulation of the very nonconservative experimental CD spectrum of the poly(rA)-GP32 complex requires that the conformation satisfies two criteria: (1) a considerable tilt of the bases (? – 10°) in combination with (2) a small rotation per base (?20°) and/or a position of the bases close to the helix axis (dx ? 0 Å). Such conformations can also explain the observed hyperchromism upon binding of GP32 to poly(rA)/(dA). Very similar structural characteristics also account for the optical properties of the complexes with GP5. These are discussed as an alternative to the structure suggested by Alma-Zeestraten for poly(dA) in the complex [N. C. M. Alma-Zeestraten (1982) Doctoral thesis Catholic University, Nijmegen, The Netherlands]. The secondary structure proposed in this work can be reconciled with the overall dimensions of the complex, assuming that the polynucleotide helix is further organized in a superhelix.     

Conformation of horse heart ferricytochrome c. III. Comparative optical rotatory dispersion study of the protein with its derivative heme undecapeptide

The optical rotatory dispersion of horse heart ferricytochrome c and of a ferri heme undecapeptide have been determined under various conditions. Analysis of the Soret region makes it possible to characterize three different states of ferricytochrome c. the native state (superposition of a negative and a positive Cotton effect); an intermediate state (single positive Cotton effect whose magnitude Δ[M] is equal to 55,000); a denatured state (single positive Cotton effect whose magnitude Δ[M] is equal to 115,000) in which compared to both the native and intermediate states a more or less important decrease in helix content is observed. The optical rotatory dispersion spectra of the Soret region of the monomeric ferri heme undecapeptide is similar to that of denatured ferricytochrome c. The multiplicity of Cotton effects observed under certain conditions for the hemopeptide is a consequence, resulting from a polymerization, of intermolecular interactions. The comparison of the optical rotatory dispersion spectra of ferricytochrome c and the ferri heme undecapeptide indicates that in the intermediate state interactions remain between the heme group and the portion of the poly pep tide chain absent in the hemopeptide. These interactions disappear in the denatured state.     

Temperature dependence of the electronic absorption spectra of polynucleotides and their components]

The temperature dependence of UV-absorption spectra of solutions nucleic bases, nucleosides, nucleotides and metilated bases, uncapable of tautomerization has been studied. The nature of such dependence, its connection with hypochromic effect is discussed. It is shown that for some methods of investigating polynucleotides it is necessary to take into account the temperature changes spectra of monomers.     

p10, a low molecular weight single-stranded nucleic acid binding protein of murine leukemia retroviruses, shows stacking interactions of its single tryptophan residue with nucleotide bases

Room temperature fluorescence and low-temperature phosphorescence studies of the association of p10, a basic low molecular weight single-stranded DNA binding protein isolated from murine leukemia viruses, point to the involvement of its single tryptophan residue in a close-range interaction with single-stranded polynucleotides. Optically detected triplet-state magnetic resonance (ODMR) techniques applied to the complex of p10 protein with the heavy atom derivatized polynucleotide poly(5-HgU) demonstrate the occurrence of stacking interactions of Trp35 with nucleic acid bases, thus agreeing with earlier reports that this residue is involved in the binding process [Karpel, R. L., Henderson, L. E., & Oroszlan, S. (1987) J. Biol. Chem. 262, 4961-4967].