Associate Hydrations and Energies of Nucleotide Bases as Revealed by Low-Temperature Field Ionization Mass-Spectrometric Data

Abstract

The formation of water clusters, polyhydrates of nucleotide bases and their associates during simultaneous condensation of water and base molecules in vacuo onto a surface of a needle emitter cooled to 170 K was studied by field ionization mass spectrometry. It was found that different emitter temperatures are characterized by a specific distribution of intensities of cluster currents, depending on the number of water molecules in clusters. These distributions correlate with structural peculiarities and the relative energetics of formation of water clusters, polyhydrates of nucleotide bases and their associates at low temperature. The features observed in mass spectra for clusters m⁹Ade (H₂O)₅, m¹Ura (H₂O)₄ and m⁹Ade m¹Ura (H₂O)₂ are treated as a result of formation of energetically favorable structures stabilized by H-bonded bridges of water molecules.

The relative association constants and formation enthalpies of the noncomplementary pairs Ade Cyt, Gua Ura and the associates which model the aminoacid-base complexes m¹Ura Gin and m₂ ^1,3Thy Gin were determined from the temperature dependencies of the intensities of mass spectra peaks in the range 290–320 K.     

Monte Carlo Simulation of Hydration of the Nucleic Acid Fragments

Abstract

Systems containing a base or a base pair and 25 water molecules, as well as a helical stack and 30 water molecules per base pair, have been simulated. Changes in the base hydration shell structure, after the bases have been included into the pair and then into the base pair stack are discussed. Hydration shells of several configurations of the base pair stacks are discussed. Probabilities of formation of the hydrogen-bonded bridges of 1, 2 and 3 water molecules between hydrophilic centres have been estimated. The hydration shell structure was shown to depend on the nature of the base pair and on the stack configuration, while dependence of the global hydration shell characteristics on the stack configuration has been proved to be rather slight. The most typical structural elements of hydration shells, in the glycosidic (minor in B-like conformation) and non-glycosidic (major) grooves, for different configurations of AU and GC stacks, have been found and discussed. The number of hydrogen bonds between water molecules and bases per water molecule was shown to change upon transformation of the stack from A to B configuration. This result is discussed in connection with the reasons for B to A conformational transition and the concept of “water economy”. Hydration shell patterns of NH₂-groups of AU and GC helical stacks differ significantly.     

Associate hydrations and energies of nucleotide bases as revealed by low-temperature field ionization mass-spectrometric data

The formation of water clusters, polyhydrates of nucleotide bases and their associates during simultaneous condensation of water and base molecules in vacuo onto a surface of a needle emitter cooled to 170 K was studied by field ionization mass spectrometry. It was found that different emitter temperatures are characterized by a specific distribution of intensities of cluster currents, depending on the number of water molecules in clusters. These distributions correlate with structural peculiarities and the relative energetics of formation of water clusters, polyhydrates of nucleotide bases and their associates at low temperature. The features observed in mass spectra for clusters m9Ade (H2O)5, m1Ura (H2O)4 and m9Ade m1Ura (H2O)2 are treated as a result of formation of energetically favorable structures stabilized by H-bonded bridges of water molecules. The relative association constants and formation enthalpies of the noncomplementary pairs Ade Cyt, Gua Ura and the associates which model the aminoacid-base complexes m1Ura Gln and m1.3(2)Thy Gln were determined from the temperature dependencies of the intensities of mass spectra peaks in the range 290-320 K.     

The meaning of the genetic code: reconstruction of the stage of prebiologic evolution

According to a certain order in sets of the two first codon bases, 20 common amino acids can be divided into 5 families each containing 4 amino acids; the corresponding order in the distribution of codon bases can be easily detected, if common amino acids are distributed for the numbers of hydrogen atoms per molecule (Sukhodolets, 1980). In the present paper, the order in the distribution of codon bases is explained on the basis of the hypothesis claiming the prebiological existence of crystalline associates composed of amino acids and bases as free molecules. In these heterogeneous crystalline associates amino acids were analogs to the base douplets and the arrangement of molecules followed a certain rule, namely: 40 protons per molecular complex forming a standard structural compartment. It is proposed that the crystalline associates existed as lyotropic liquid crystals with hydrocarbons as solvent. The genetical code allows to discover two different original crystallization types for bases and amino acids. Therefore, the life possibly originates from combining in the same structure different crystallization patterns, which resulted in formation of a finite crystalline associate.     

The effects of chain length on the secondary structure of oligoadenylates

The oligoadenylates (Ap)_2–4A have been studied by proton magnetic resonance (pmr) spectroscopy. All the exterior base protons and a number of the interior base proton resonance have been assigned. The results of this work showed that the adenine bases in these oligoadenylates are intramolecularly stacked at 20°C with their bases oriented preferentially in the anti conformation about their respective glycosidic bonds. The oligomers were found to associate extensively even at concentrations of 0.02 M, primarily via “end-to-end” stacking. With increasing temperature, the oligomer bases destack, but it is argued that this unfolding process cannot be described in terms of a two-state stacked versus unstacked model. Instead, the temperature dependences of the base proton chemical shifts support a base-oscillation model. The relationship between this model and the two-state model is discussed. Finally, on the basis of the chain-length dependence of the proton chemical shifts of the various adenine bases, it was concluded that subtle variations in the secondary structure of these oligomers exist with increasing chain length. Evidence is presented to show that the effects of distant base shielding are considerably smaller than what was previously estimated. The observed departures from the “extended dimer” model are attributed to differences in the relative orientations of the bases with respect to their neighbors in the oligomer.     

Effect of detergent on aggregation of the light-harvesting chlorophyll a/b complex of photosystem 2 and its impact for carotenoid function and fluorescence quenching

Spectroscopy was used to investigate the fluorescence quenching mechanism in light-harvesting complex 2 (LHC2). The 77 K fluorescence excitation spectroscopy was performed for detection of aggregation state of LHC2 treated with different concentrations of octylphenol poly(ethyleneglycol ether)₁₀ (TX-100). Resonance Raman (RR) spectra excited with 488, 496, and 514 nm provided molecular configuration of neoxanthin, lutein 1, and lutein 2, respectively. At increased concentration of TX-100, the RR signals of xanthophylls were enhanced in the four frequency regions, which was accompanied with increase of fluorescence of chlorophyll (Chl) a. Thus the absorption of the three xanthophyll molecules was inclined to excitation wavelength, which proved that functional configurations of xanthophyll molecules in LHC2 were vital for fast transfer of excitation energy to Chl a molecules. Changes in the v4 region (C-H out-of-plane bending modes, at ∼960 cm⁻¹ in RR spectra) demonstrated that the twist feature of neoxanthin, lutein 1, and lutein 2 molecules existed in LHC2 trimers, however, it was lost in the LHC2 macro-aggregates. In the second derivative absorption spectra of LHC2, neoxanthin absorption was not detected in LHC2 macro-aggregates, while evident absorption was found in LHC2 trimers and this absorption decreased obviously when TX-100 concentration was higher than 1 mM. Hence the neoxanthin molecule had a structural role in formation of LHC2 trimers. The RR and absorption spectra also implied that carotenoid molecules constructed the functional LHC2 trimers via their intrinsic configuration features, which enabled energy transfer to Chl a efficiently and led to lower fluorescence quenching efficiency. In contrast, these intrinsic twist configurations were lost in LHC2 macro-aggregates and led to lower energy transfer efficiency and higher fluorescence quenching efficiency.     

Binding of Net-Fla,a Netropsin-Flavin Hybrid Molecule,to DNA: Molecular Mechanics and Dynamics Studies in vacuo and in Water Solution

Abstract

We have studied the binding of the hybrid netropsin-flavin (Net-Fla) molecule onto four sequences containing four A.T base pairs. Molecular mechanics minimizations in vacuo show numerous minimal conformations separated by one base pair. 400 ps molecular dynamics simulations in vacuo have been performed using the lowest minima as the starting conformations. During these simulations, the flavin moiety of the drug makes two hydrogen bonds with an amino group of a neighboring guanine. A 200 ps molecular dynamics simulation in explicit water solution suggests that the binding of Net-Fla upon the DNA substrate is enhanced by water bridges. A water molecule bridging the amidinium of Net-Fla to the N3 atom of an adenine seems to be stuck in the dmg-DNA complex during the whole simulation. The fluctuations of the DNA helical parameters and of the torsion angles of the sugar-phosphate backbone are very similar in the simulations in vacuo and in water. The time auto-correlation functions for the DNA helical parameters decrease rapidly in the picosecond range in vacuo. The same functions computed from the water solution molecular dynamics simulations seem to have two modes: the rapid mode is similar to the behavior in vacuo, and is followed by a slower mode in the 10 ps range.     

Circular dichroism of polynucleotides: dimers as a function of conformation

Working within the restrictions of a model, we have calculated the circular dichroism of the dinucleoside phosphates ApA, CpC, and CpA for various conformations. Comparing the calculated curves with those measured in aqueous solution we find agreement for (1) ApA as a right-handed helix with both bases either as in B-form DNA, or else rotated 180° around the glycosidic bond, (2) CpC as the right-handed conformation with both bases as in DNA, (3) ApC as either the right-handed conformation with both bases as in DNA, or else as a left-handed helix with both bases rotated 180°, and (4) CpA as either a left-handed helix with both bases in a left-handed DNA, or else in the right-handed conformation with both bases rotated 180°. In addition, we have investigated circular dichroism as a measure of unstacking. We find that opening the bases to a 90° total angle (base planes perpendicular) reduces the intensity of the calculated bands to 20% of their original value. Further, we find that allowing the sliding of one base past the other does not lead to a temperature dependence consistent with experiment.     

DNA and RNA oligomer thermodynamics: The effect of mismatched bases on double-helix stability

The thermodynamic parameters for the double-strand formation of the molecules rCAmG + rCU_nG, m,n = 5–7, and dCA_mG + dCT_nG, m,n = 5,6, were measured from optical melting curves. Normal helices are formed when m = n. The deoxyoligomers are more stable than the ribo-oligomers, due to a more favorable enthalpy. Double helices with mismatched bases can be formed by mixing oligomers with m ≠ n. Such helices may form several possible structures. A structure with a dangling base is favored over a structure with a bulged base. The destabilization of the double strands by the formation of a bulged base was determined to be greater than 1.6 kcal/mol at 10°C. The extent of aggregation in the oligomer double strand rCA₇G·rCU₇G was determined using ultracentrifugation equilibrium. The possible effects of aggregation on the determination of the thermodynamic parameters for double-strand formation are discussed.     

Variable Temperature Infrared Spectroscopy of Cytosine-Guanine Base Pairs: Tautomerism Versus Polarization

Abstract

This report describes an infrared (IR) spectroscopic study of a model cytosine - guanine base pair. This base pair is part of a self-consistent experimental system based on lipophilic ribose derivatives of cytidine (C), guanosine (G) and O⁶-methylguanosine (O⁶MeG) that are soluble in non-aqueous, low dielectric solvents at appreciable concentrations. Previous experiments on this system have revealed different rotation dynamics for the amino bonds within the CG base pair, an observation that could be explained by the presence of rare tautomers (P.O. Lowdin, Reviews of Modern Physics 35, 724 (1963)), or by mutual polarization of the base pairs (L.D. Williams, N.G. Williams and B.R. Shaw, J. Am. Chem. Soc. 112, 829 (1990)). The IR spectra in the OH and NH stretching region indicate formation of hydrogen-bonded CG base pairs and self associates in 1,2-dichlorobenzene over a temperature range from 10 to 290K. Changes in the lineshapes and intensities of the IR bands with temperature correlate with phase transitions of the solvent but no evidence is seen for an OH stretching band that would indicate the formation of hydroxyl tautomers within base pairs. Similarly, the relative intensities of the C=O stretching bands of CG in cyclohexane solution remain constant over this same temperature range, confirming that within the base pair, the tautomeric states of the bases remain essentially unperturbed in the 2-amino/6-keto form of G and the 2-keto/4-amino form of C. The spectra of O⁶-MeG aid in the band assignments, since this molecule is frozen in an equivalent of the 2-amino/6-hydroxyl tautomer, but without the OH group and its associated stretching band. We conclude that the probability of tautomerism does not appear to be sufficient to explain the different rotation dynamics for the two amino bonds of the CG base pair. Rather it is argued that mutual polarization within the base pair, which would increase the bond order of the amino bond of C within the base pair, can explain the results without the formation of unconventional tautomers.     

Simulations of the solvent structure for macromolecules. I. Solvation of B-DNA double helix at T = 300 K

Monte Carlo simulations are reported for a system of 447 water molecules enclosing a B-DNA double-helix fragment with 12 base pairs and the corresponding sugar and phosphate units. From a detailed analysis on the interaction energies and probability distributions (at a simulated temperature of 300 K), the water molecules can be partitioned into clusters strongly interacting with (1) the phosphates, (2) the sugars, (3) the sugars and the bases, and (4) the base pairs. In addition, transgroove and interphosphate filament of hydrogen-bonded water molecules have been detected. From simulations performed with variable numbers of water molecules, a theoretical isotherm has been obtained, with the characteristic sigmoidal shape, known from absorption–desorption experiments on related systems. The expected main features for the structure of water molecules solvating B-DNA with Na⁺ counterions are briefly discussed at the end of the paper.     

Effect of Endogenous and Exogenous Urate on the Uricase Formation by Streptomyces sp.

Cells of a strain of Streptomyces sp. were incubated with an equivalent quantity of urate, xanthine, 6,8-dihydroxypurine or hypoxanthine in a medium deprived of other nitrogen source. The amount of uricase produced by these cells was shown to differ significantly, increasing in the following order of purine bases added to the medium: urate, xanthine, 6,8-dihydroxypurine and hypoxanthine. Of these was only urate indicated to be the inducer of uricase formation, and the difference in the quantity of uricase produced was found to be based on the duration of enzyme formation. The rate of uricase formation was essentially identical regardless of the purine bases supplied to cells.

Allantoin was accumulated in medium in remarkably different manners depending on the purine bases, which suggested the diversity in the mode of generation of urate in cells. Urate was generated at the slowest rate in the cells incubated with hypoxanthine, although the largest amount of uricase was produced, However, urate supplied to cells at the same rate but from medium failed to support the enzyme formation when the activity increased to a certain level. In order that the same amount of uricase was produced by the cells incubated with the different purine bases, the initial concentration of the purine bases should be raised so that they could remain in medium for the same incubation time.

Intracellular compartmentalization that might segregate endogenous and exogenous urate and might cause the difference in “effeciency” of these urate molecules as the inducer of uricase formation has been discussed.     

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.     

Targeted conservative formation of cointegrates between two DNA molecules containing IS26 occurs via strand exchange at either IS end

We recently proposed a model for targeted, conservative cointegrate formation between DNA molecules each containing a copy of IS26, that involves Tnp26‐catalyzed strand exchange occurring at either the two left ends or the two right ends of the IS. Here, this model was validated by altering the bases at the outer left terminus, right terminus or both termini of one IS26. The correct bases at both ends were required in the untargeted replicative mode. However, when only one end was altered in one participating IS the frequency of targeted, conservative cointegrate formation was not reduced. The distribution of the altered bases in the cointegrates confirmed that the reaction occurred at the end where the terminal bases of both IS were correct, and cointegrates were not formed when both ends of the same IS were altered. The terminal bases of the active IS26 were also required to support deletion of the aphA1a translocatable unit (TU) from Tn4352B. The choices made by an incoming TU with a wild‐type IS26 when the target plasmid included one wild‐type IS26 and one with a frameshift in tnp26 demonstrated that Tnp26 exhibits a strong preference for cis action.     

Influence of growth phase and zeolite clinoptilolite on the concentration of sphingoid bases in Saccharomyces uvarum brewer’s yeast

Sphingolipids having a long-chain sphingoid base backbone are primarily located in the yeast’s plasma membrane. They are found in various types of foods, and although they are not essential food ingredients, they play an important role as bioactive molecules in preventing certain human diseases. Today, due to its high nutritional value, brewer’s yeast is increasingly being used in the food and pharmaceutical industry. The aim of this study was to evaluate the potential of S. uvarum, a by-product of the brewing industry, as an economically feasible source of sphingolipids. For that purpose, the growth phase dependence on sphingolipid production in S. uvarum as well as the effect of zeolite addition to the growth medium was investigated. The experiments were designed to explore the dependence of growth phase on sphingolipids metabolism, by comparing initial (starter) culture of brewer’s yeast (laboratory propagated, designated as zero yeast generation, serving here as control), and surplus brewer’s yeast (a residue produced after 5 successive beer fermentations), by-product of beer fermentation, with and without the addition of zeolite. HPLC analysis of individual molecular species of sphingoid bases obtained by acid hydrolysis of complex sphingolipids from S. uvarum yeast produced the following results: about 65% of total sphingoid bases represents C18 phytosphingosine, about 32% represents unknown long-chain base, and about 1.5–2% represents C18 DL-erythro-sphinganine. In the case of C18 phytosphingosine, production was about 11.5-fold higher during exponential phase compared with the other growth phases. For C18 DL-erythro-sphinganine, production was highest during the lag and acceleration phase of growth. In most cases, zeolite addition (1%) to the growth medium resulted in an increase up to 2.5-fold in the sphingoid bases level.     

Vacuum ultraviolet absorption spectra of sublimed films of nucleic acid bases

Measurements of the optical absorption spectra of the sublimed films of adenine, guanine, cytosine, thymine, and uracil were extended down to 120 mμ at room temperature. Several remarkable absorption peaks were found to exist below 190 mμ in addition to the already known ones near 260 mμ and 200 mμ. The intensities of these peaks were comparable to or, in some cases, even larger than those near 260 mμ and 200 mμ. It was found that the relative intensities of the absorption peaks differed considerably from sample to sample for all five bases, probably due to variation in the arrangement of the bases in the sublimed films. It was found also that the absorption spectra of the sublimed films change with aging on standing in vacuo at room temperature. On comparing the spectra of several fresh samples it was found that the intensities of the transitions with wavelength longer than 160 mμ of adenine, guanine, and uracil films are inversely correlated with those of the transitions shorter than 160mμ.     

Structure of Three-Way DNA Junctions 1. Non-Planar DNA Geometry

Abstract

Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20–21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60–90°. Rotation of the hairpin position in the three- way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60–90° between the arms. Computer modeling of the three-way junction with 60° pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.     

Monte Carlo simulations of a protein molecule with and without hydration energy calculated by the hydration-shell model

Monte Carlo simulations of a small protein, carmbin, were carried out with and without hydration energy. The methodology presented here is characterized, as compared with the other similar simulations of proteins in solution, by two points: (1) protein conformations are treated in fixed geometry so that dihedral angles are independent variables rather than cartesian coordinates of atoms; and (2) instead of treating water molecules explicitly in the calculation, hydration energy is incorporated in the conformational energy function in the form of g _i A _i, whereA _i is the accessible surface area of an atomic groupi in a given conformation, andg _i is the free energy of hydration per unit surface area of the atomic group (i.e., hydration-shell model). Reality of this model was tested by carrying out Monte Carlo simulations for the two kinds of starting conformations, native and unfolded ones, and in the two kinds of systems,in vacuo and solution. In the simulations starting from the native conformation, the differences between the mean propertiesin vacuo and solution simulations are not very large, but their fluctuations around the mean conformation during the simulation are relatively smaller in solution thanin vacuo. On the other hand, in the simulations starting from the unfolded conformation, the molecule fluctuates much more largely in solution thanin vacuo, and the effects of taking into account the hydration energy are pronounced very much. The results suggest that the method presented in this paper is useful for the simulations of proteins in solution.     

Corolla tube formation in six species of apocynaceae

Corolla tube formation inTrachelospermum asiaticum, Nerium indicum var.leucanthum, Anodendrom affine, Vinca major, Catharanthus roseus andAmsonia elliptica was investigated anatomically. The corolla tube formation among these species is basically similar. The bases of petal primordia extend laterally to the interprimordial regions, the upward growth occurrig at those regions just beside the petal bases. The extending petal bases connect with each other at the bases of the abaxial side of stamen primordia in the early stage of the corolla development. The upward growth at the coonnected regions results in the formation of a short corolla tube but is weakened rapidly. At the stage of the mutual connection of petal bases, a common base of petal and stamen primordia is initiated. This common base develops into the lower portion of the corolla tube, i.e. the portion below the stamen insertion. In a relatively late stage, adjacent margins, of the corolla lobes fuse postgenitally at their lower portions, resulting in the formation of almost all of the upper portion of the corolla tube. The corona inNerium andVinca is initiated by the active adaxial growth of the upper portion of the corolla tube.