Electronic and spacial structure of orthophosphate and dimethylorthophosphate

The applicability of a number of semiempirical CNDO variants for the computations of the electronic structure of different conformations for all possible ionic forms of dimethylorthophosphate and orthophosphate has been considered. The Boyd-Whitehead variant of CNDO approximation (CNDO/BW) with the original parameters we chose for the P-O bond gives the best qualitative and sometimes quantitative correspondence with ab initio methods. We have utilized this approximation to compute the dependences of energies and P-O bond strengths in P-O-CH3 (P-O-H) fragments of dimethylorthophosphate and orthophosphate versus the angles of rotation of these fragments about the P-O bonds. It is shown that, during the rotation, the increase in the strength of one P-O bond is accompanied by the labiality of another one. The energy minima of dimethylorthophosphate and orthophosphate anions correspond to conformations with approximately equal strengths of the P-O bonds. Thus, none of these bond strengths achieves a minimum. The protonation of dimethylorthophosphate and orthophosphate results in a strengthening of P-O bonds and decreases the dependence of their strength on the variation of torsion angles. Di- and three-anionic derivatives of dimethylorthophosphate and orthophosphate are also discussed. It is shown that the strength of P-O bond diminishes as the negative charge of dimethylorthophosphate and orthophosphate grows. It the case of dianion, the dependence of bond rigidity on the torsion angle is less pronounced than in the case of monoanione. Our theoretical results are compared with the experimental data known from literature. The importance of our data for elucidating some essential features of functioning of enzymes accomplishing the breakdown and formation of P-O bonds is also discussed.     

Stereochemistry of nucleic acids and polynucleotides. VI. Minimum energy conformations of dimethyl phosphate

As part of a study on the conformation of polynucleotides and nucleic acids the preferred conformations of the model conpound dimethyl phosphate are worked out using potential energy functions. In calculating the total potential energy associated with the conformation, nonbonded, torsional, and electrostatic terms have been considered. The variation of the total conformational energy is represented as a function of two torsion angles ? and ψ which are the rotations about the two phosphoester bonds. The most stable conformations are found to be the gauche–gauche conformations about these bonds. The conformations observed for phosphodiesters in the solid state and in the proposed structures of polynucleotides and nucleic acids cluster around the minimum. Also, regions of minimum energy correspond well with the typical allowed regions of a representative dinucleotide.     

A conformational study of nucleic acid phosphate ester bonds using phosphorus-31 nuclear magnetic resonance.

A systematic phosphorus-31 nuclear magnetic resonance study of some nucleic acid constituents (6-N-(dimethyl)adenylyl-(3',5')-uridine and some nucleotide methyl esters) is presented. The temperature dependent phosphorus-31 chemical shifts were analyzed by standard thermodynamic procedures. It is shown that gt conformations about the P-O ester bonds have a lower free energy content relative to gg conformers.     

Conformational analysis of inulobiose by molecular mechanics

Conformational energies for inulobiose [beta-D-fructofuranosyl-(2----1)-beta-D-fructofuranoside], a model for inulin, were computed with the molecular mechanics program MMP2(85). The torsion angles of the three linkage bonds were driven in 20 degree increments, and the steric energy of all other parameters was minimized. The linkage torsion angles defined by C-1'-C-2'-O-C-1 (phi) and O-C-1-C-2-O-2 (omega) have minima at +60 degrees and -60 degrees, respectively, regardless of side group orientation; accessible minima exist at other staggered conformations. The torsion angle at the central bond C-2'-O-1-C-1-C-2 (psi) was approximately 180 degrees in all the low-energy conformers. This appears to be generally true for rings linked by three bonds. The fructofuranose rings initially had low-energy 4/3T conformations (angle of pseudorotation, phi 2 = 265 degrees) that were retained except when the linkage conformations created severe inter-residue conflicts. In those cases, almost all puckerings of the furanose rings were found.     

Structure and Dynamics of Double Helical DNA in Torsion Angle Hyperspace: A Molecular Mechanics Approach

Abstract

Analysis of the conformational space populated by the torsion angles and the correlation between the conformational energy and the sequence of DNA are important for fully understanding DNA structure and function. Presence of seven variable torsion angles about single covalent bonds in DNA main chain puts a big challenge for such analysis. We have carried out restrained energy minimization studies for four representative dinucleosides, namely d(ApA):d(TpT), d(CpG):d(CpG), d(GpC):d(GpC) and d(CpA):d(TpG) to determine the energy hyperspace of DNA in context to the values of the torsion angles and the structural properties of the DNA conformations populating the favorable regions of this energy hyperspace. The torsion angles were manipulated by constraining their values at the reference points and then performing energy minimization. The energy minima obtained on the potential energy contour plots mostly correspond to the conformations populated in crystal structures of DNA. Some novel favorable conformations that are not present in crystal structure data are also found. The plots also suggest few low energy routes for conformational transitions or the associated energy barrier heights. Analyses of base pairing and stacking possibility reveal structural changes accompanying these transitions as well as the flexibility of different base steps towards variations in different torsion angles.     

Stereochemical effects of non-ionic methylphosphonates on nucleotide conformations.

The preferred conformations of deoxyribo and ribonucleoside 3'-methylphosphonates are analysed by minimizing the conformational energy as a function of all the major parameters including the sugar ring for both the S- and R-isomers. The results show that neither the substitution nor the nature of the diastereomer affects significantly the preferred conformations compared to the naturally occurring nucleoside 3'-phosphates. The preferred range of C3'-O3' bond torsions or the phase angles of pseudorotation (P) of the sugar are unaffected. The chiral substitution on the phosphate always adopts a conformation distal to the secondary C3' carbon atom in the minimum energy conformational state. Further, it introduces certain restrictions on the preferred range of P-O3' torsions depending on the methylphosphonate configuration. Methylphosphonate, especially the S-isomer, renders the normal gauche- range of P-O3' bond torsions responsible for the stacked helical duplexes to be energetically unfavourable besides introducing a high energy barrier between trans and gauche conformations. Therefore it is suggested that duplexes with S-methylphosphonate may favour extended phosphodiester conformations. These factors explain the observed lower melting temperature as well as the downfield shifts in the 31P signals in duplexes containing the S-isomer.     

Analytical determination of orthophosphate in water

Methods for orthophosphate determination and the problems of interferences are reviewed.An important group of methods utilizes the phosphomolybdate complex. The complexation step, the reduction step and the extraction step are treated separately and alternative procedures compared.Another group of methods uses ion association complexes; they are primarily used in physiology and not commonly used in water analyses today.Enzymatic methods for orthophosphate analysis in natural waters have been developed lately and are ready for application in selected waterbodies.Flame spectroscopic, fluorometric, gas chromatographic, ion exclusion chromatographic, inductively coupled plasma and other methods are also shortly presented.Radiobiological bioassays for orthophosphate are also available.In conclusion it was emphasized that the most common and reliable technique still is the molybdenum blue method as modified by Murphy & Riley (1962).The need for more specific and sensitive methods is particularly strong in investigations of phosphorus turnover and phosphorus limitation in natural waters. For these purposes the enzymatic phosphatase methods has advantages due to their specificity for orthophosphate and they might offer an alternative to the molybdenum blue method.     

Vibrational analysis of nucleic acids. V. Force field and conformation-dependent modes of the phosphodiester backbone modeled by diethyl phosphate.

A generalized valence force field is derived for the diethyl phosphate anion [(CH3CH2O)2PO2-] and its deuterium [(CH3CD2O)2PO2-, (CD3CH2O)2PO2- and (CD3CD2O)2PO2-] and carbon-13 [(CH3 13CH2O)2PO2-] derivatives in the stable trans-gauche-gauche-trans conformation. Normal coordinate analysis of the trans-gauche-gauche-trans conformer, which serves as a structural analog of the nucleic acid phosphodiester group, is based on comprehensive infrared and Raman spectroscopic data and vibrational assignments obtained for the diethyl phosphate anion. The generalized valence force field is in good agreement with the scaled ab initio force field of diethyl phosphate and represents significant improvement over earlier modeling of the phosphodiester moiety with dimethyl phosphate. The conformational dependence of skeletal C-C-O-P(O2-)-O-C-C stretching vibrations is also explored. Starting with the trans-gauche-gauche-trans conformation, the frequency dependence of skeletal stretching modes has been obtained by stepwise rotation of the torsion angles of the P-O and C-O bonds corresponding to nucleic acid torsions alpha (P-O5'), beta (O5'-C5'), epsilon (C3'-O3'), and zeta (O3'-P). Both symmetric and antisymmetric phosphoester stretching modes are highly sensitive to P-O and C-O torsions, whereas symmetric and antisymmetric phosphodioxy (PO2-) stretching modes are less sensitive. The present results provide an improved structural basis for understanding previously developed empirical correlations between vibrational marker bands and nucleic acid backbone conformation.     

UV Photoelectron Spectroscopy and Ab Initio Characterization of Valence Orbital Structures and Conformations of Neutral Phosphate Esters

Abstract

The Hel UV photoelectron spectrum of trimethyl phosphate (TMP) has been measured and interpreted with the aid of SCF molecular orbital calculations carried out with STO-3G, STO-3G* and 4–31G basis functions. The photoelectron spectrum of TMP is more accurately reproduced by results from 4–31G calculations than by results from STO-3G or STO-3G* calculations. However, all three basis sets yield results which predict the same assignment of the photoelectron spectrum. Results at the 4–31G level indicate that whether calculations are based on crystallographic bond angles and bond lengths or on STO-3G optimized geometries has little effect on the energetic ordering of the upper occupied orbitals. The energetic ordering of orbitals is also found to be only weakly dependent upon the torsional angle φ, describing rotation of ester groups about P-O bonds and upon the torsional angle ψ, describing rotation of methyl groups about C-O bonds. For trimethyl phosphate, with C₃ symmetry, the vertical ionization potentials of the upper occupied orbitals are 10.81 eV (8e), 11.4 eV (9a), 11.93 eV (7e), 12.6–12.9 eV (8a and 6e), 14.4 eV (7a) and 15.0–16.0 eV(5e and 6a). Calculations at the 4–31G level indicate that many of the highest occupied orbitals in neutral dimethyl phosphate and methyl phosphate have energies and electron distributions similar to orbitals in TMP.

For TMP, a search for optimized values of φ and ψ has been carried out at the STO-3G* level. In agreement with previous NMR studies and with classical potential calculations, the STO- 3G* results indicate that both the gauche φ= 53.1 °) and anticlinal (φ = 141.9°) conformations are thermally accessible. Also in agreement with the classical potential calculations, the STO-3G* results predict that in the all gauche conformation energy is minimized when the methyl groups assume a staggered geometry (ψ= 60° to 80°) and that an energy maximum occurs for an eclipsed geometry (ψ = 0° to 20°). A study of the dependence of optimized values of O-P-O ester bond angles on the torsional angles, φ, was carried out at the STO-3G, STO-3G* and 4–31G levels. The results demonstrate that for C₃ symmetry, the coupling of O-P-O angles to φ is influenced by repulsive steric interactions.     

Theoretical conformational analysis of methylamide of N-acetyl-L-lysine]

The spatial structure of the methylamide of N-acetyl-L-lysine has been analysed taking into account non-bonded and electrostatic interactions, torsional energy, bond angles distortion and hydrogen bonding. Conformational capacities of the backbone and mutual dependence of spatial structures of the backbone and the side chain was described by conformational maps obtained by energy minimisation, the dihedral angles and the bond angles of the side chain being varied for every phi, psi point. Every possible combination for phi, psi, x1-x5-angles was used corresponding to the stable form of the backbone and to torsion potential minima of the initial approximations in the calculation of preferred conformations of the molecule. Comparisons are made between stable forms of the methylamide of N-acetyl-L-lysine and Lys residues in proteins with known structure.     

Effect of side chains on the conformational energy and rotational strength of the n-pi transition for some alpha-helical poly-alpha-amino acids

Calculations of the dependence of the conformational energy and the rotational strength of the amide n–π* electronic transition (in a series of α-helical polyhel-α- amino acids with different side chains) on conformation have been carried out. The conformational energies were computed by procedures developed in this laboratory; the computation of rotational strengths was carried out by the method of Schellman and Oriel, with a slight modification. Polyamino acids with both nonpolar and polar side chains were considered; in the latter case, it was assumed that the only influence of the polar side chain was on the backbone conformation and on the electrostatic field which perturbs the amide chromophore of the backbone. Only conformations in the range of backbone dihedral angles of the right- and left-handed a-helices were considered, and the assumption of regularity (i.e., uniformity of dihedral angles in every residue) was made. The rotational strength per residue was found to vary markedly with chain length (in oligomers of up to 40 residues long); both the conformational energy per residue and the rotational strength per residue were found to vary significantly with the backbone conformation, which in turn depends on the nature of the side chain. The geometry of the hydrogen bond in the α-helical backbone is the most important factor which influences the dependence of the rotational strength on conformation. The implications of these results, for the interpretation of experimental circular dichroism and optical rotatory dispersion data, are discussed.     

UV photoelectron spectroscopy and ab initio characterization of valence orbital structures and conformations of neutral phosphate esters

The HeI UV photoelectron spectrum of trimethyl phosphate (TMP) has been measured and interpreted with the aid of SCF molecular orbital calculations carried out with STO-3G, STO-3G* and 4-31G basis functions. The photoelectron spectrum of TMP is more accurately reproduced by results from 4-31G calculations than by results from STO-3G or STO-3G* calculations. However, all three basis sets yield results which predict the same assignment of the photoelectron spectrum. Results at the 4-31G level indicate that whether calculations are based on crystallographic bond angles and bond lengths or on STO-3G optimized geometries has little effect on the energetic ordering of the upper occupied orbitals. The energetic ordering of orbitals is also found to be only weakly dependent upon the torsional angle phi, describing rotation of ester groups about P-O bonds and upon the torsional angle psi, describing rotation of methyl groups about C-O bonds. For trimethyl phosphate, with C3 symmetry, the vertical ionization potentials of the upper occupied orbitals are 10.81 eV (8e), 11.4 eV (9a), 11.93 eV (7e), 12.6-12.9 eV (8a and 6e), 14.4 eV (7a) and 15.0-16.0 eV (5e and 6a). Calculations at the 4-31G level indicate that many of the highest occupied orbitals in neutral dimethyl phosphate and methyl phosphate have energies and electron distributions similar to orbitals in TMP. For TMP, a search for optimized values of phi and psi has been carried out at the STO-3G*level. In agreement with previous NMR studies and with classical potential calculations, the STO-3G* results indicate that both the gauche (phi = 53.1 degrees) and anticlinal (phi = 141.9 degrees) conformations are thermally accessible. Also in agreement with the classical potential calculations, the STO-3G* results predict that in the all gauche conformation energy is minimized when the methyl groups assume a staggered geometry (psi = 60 degrees to 80 degrees) and that an energy maximum occurs for an eclipsed geometry (phi = 0 degrees to 20 degrees). A study of the dependence of optimized values of O-P-O ester bond angles on the torsional angles, phi, was carried out at the STO-3G, STO-3G* and 4-31G levels. The results demonstrate that for C3 symmetry, the coupling of O-P-O angles to phi is influence by repulsive steric interactions.     

The role of carbon-donor hydrogen bonds in stabilizing tryptophan conformations

Although most side-chain torsion angles correspond to low-energy rotameric positions, deviations occur with significant frequency. One striking example arises in Trp residues, which have an important role in stabilizing protein structures because of their size and mixed hydrophobic/hydrophilic character. Ten percent of Trp side-chains have unexplained conformations with chi(2) near 0 degrees instead of the expected 90 degrees. The current work is a structural and energetic analysis of these conformations. It is shown that many Trp residues with these orientations are stabilized by three-center carbon-donor hydrogen bonds of the form C-H...X...H-C, where X is a polar hydrogen-bond acceptor in the environment of the side-chain. The bridging hydrogen bonds occur both within the Trp side-chain and between the side-chain and the local protein backbone. Free energy maps of an isolated Trp residue in an explicit water environment show a minimum corresponding to the off-rotamer peak observed in the crystallographic data. Bridging carbon-donor hydrogen bonds are also shown to stabilize on-rotamer Trp conformations, and similar bridging hydrogen bonds also stabilize some off-rotamer Asp conformations. The present results suggest a previously unrecognized role for three-center carbon-donor hydrogen bonds in protein structures and support the view that the off-rotamer Trp side-chain orientations are real rather than artifacts of crystallographic refinements. Certain of the off-rotamer Trp conformations appear to have a functional role.     

Boltzmann-type distribution of side-chain conformation in proteins

We analyze packing imperfections in globular proteins as reflected in deviations of torsion angles from the equilibrium values for the isolated side chains. The distribution of conformations of methionine and lysine residues in a database of high-resolution structures is compared with energies of model compounds calculated with high-level quantum-mechanics. The distribution of the C-C and C-S torsion angles (chi(3)) correlates well with the Boltzmann factor of the torsion energy, exp(-betaE) of the model compounds C(2)H(5)-C(2)H(5) and C(2)H(5)-S-CH(3). An exponential relation was again found between the relative occurrence of g+, g- and t conformations for C(alpha)-C(beta) bonds in long side chains and the energy differences of rotamers of alpha-amino n-butyric acid, when dependence on backbone conformation was taken into account. The distribution of all 27 rotamers of methionine was correlated with the energy differences between the model's rotamers, corrected for clashes with nearby residues, the correlation being good for a set with backbone in the beta-conformation, but less clear for backbone alpha-conformation. In all correlations, the value of the coefficient beta corresponds to a temperature of circa 300 K. These results can be interpreted with a model that considers the structure of a folded protein as resulting from packing imperfectly complementary parts, with a requirement of an overall low energy. Compromises are required to optimize the fit of nonbonded contacts with surrounding groups, and side chains assume conformations away from the energy minimum. An exponential distribution is a most probable distribution, and this can be established easily under conditions other than thermal equilibrium.     

Geometry of the five-membered ring mathematical demonstration of the pseudorotation formulae

The geometrical relations between the 15 typical parameters (bond lengths and angles, torsion angles) of a five-membered ring are derived for any ring then for a regular one. It is demonstrated that for the case of the 20 symmetrical C ₂ and C _sconformations, only geometrical considerations are needed to obtain the pseudorotation formulae for the torsion angles. However, the puckering intensity as well as the bond angle values cannot be expressed from geometrical constraints alone but would require energetical considerations.     

Stereochemistry of ATP and GTP bound to fish haemoglobins. A transferred nuclear overhauser enhancement, 31P-nuclear magnetic resonance, oxygen equilibrium and molecular modelling study

This study was undertaken in order to test the models of ATP and GTP binding to carp deoxyhaemoglobin proposed by Perutz & Brunori (1982) and to find out why GTP is a more potent allosteric effector than ATP. We have determined the conformations of both nucleoside triphosphates by nuclear magnetic resonance studies and found them to be the same. The purines are in anti conformation about the glycosidic bond that links them to the ribose; the pentose ring is 3'-endo; the P-O5'-C5'-C4' torsion angle lies in the trans domain (180 degrees +/- 20 degrees); the P alpha-O-P beta and P beta-O-P gamma angles are as in the free nucleotides, i.e. the trinucleotide chain is fully extended. Models having this conformation were fitted, first manually and then by energy refinement, to the effector site of an atomic model of human deoxyhaemoglobin in which the side-chains in the NA, EF and H segments had been replaced by those of carp. The results showed the location of the polar groups in carp haemoglobin to be such that (PO4) gamma can accept hydrogen bonds from Val NA1 beta 2 and from Arg H21 beta 1, while (PO4) beta and (PO4) alpha can accept hydrogen bonds from Lys EF6 beta 1 and beta 2. In ATP, the 6-amino group of the purine can donate a hydrogen bond to Glu NA2 beta 1. In GTP, the 2-amino group can donate a hydrogen bond to Glu NA2 beta 1; in addition, Val Na1 beta 1 can donate a hydrogen bond to O2' of the ribose. This additional hydrogen bond may explain why in carp haemoglobin GTP is a stronger allosteric effector than ATP. We have found the influence of the two allosteric effectors on the oxygen affinity of trout IV haemoglobin to be the same, even though the only difference in the lining of the allosteric effector sites lies in the replacement of Glu Na2 beta in carp by Asp in trout IV haemoglobin. Model building then showed that formation of a hydrogen bond between Asp Na2 beta and the 2-amino group of guanine precludes formation of a hydrogen bond between Val NA1 beta and O2' of the ribose or vice versa, which makes the number of hydrogen bonds formed between trout IV haemoglobin and GTP the same as those formed with ATP.     

Flexibility of 3′,5′ Deoxyribonucleooside Diphosphates

Abstract

In 3′,5′ deoxyribonucleoside diphosphates, in addition to the nature of the base and the sugar puckering, there are six single bond rotations. However, from the analysis of crystal structure data on the constituents of nucleic acids, only three rotational angles, that are about glycosyl bond, about C4′-C5′ and about C3′-O3′ bonds, are flexible. For a given sugar puckering and a base, potential energy calculations using non-bonded, electrostatic and torsional functions were carried out by varying the three torsion angles. The energies are represented as isopotential energy surfaces. Since the availability of the real-time color graphics, it is possible to analyse these isopotential energy surfaces. The calculations were carried out for C3′ exo and C3′ endo puckerings for deoxyribose and also for four bases. These calculations throw more light not only on the allowed regions for the three rotational angles but also on the relationships among them. The dependence of base and the puckering of the sugar on these rotational angles and thereby the flexibility of the 3′,5′ deoxyribonucleoside diphosphates is discussed. From our calculations, it is now possible to follow minimum energy path for interconversion among various conformers.     

Correlation between the backbone and side chain conformations in 5′-nucleotides. The concept of a ‘rigid’ nucleotide conformation

Conformational energies of the 5′-adenosine monophosphate have been computed as a function of χ and ψ, of the torsion angles about the side-chain glycosyl C(1′)–N(9) and of the main-chain exocyclic C(4′)–C(5′) bonds by considering nonbonded, torsion, and electrostatic interactions. The two primary modes of sugar puckering, namely, C(2′)-endo and C(3′)-endo have been considered. The results indicate that there is a striking correlation between the conformations about the side-chain glyocsyl bond and the backbone C(4′)–C(5′) bond of the nucleotide unit. It is found that the anti and the Gauche–Gauche (gg), conformations about the glycosyl and the C(4′)–C(5′) bonds, respectively, are energetically the most favored conformations for 5′-adenine nucleotide irrespective of whether the puckering of the ribose is C(2′)-endo or C(3′)-endo. Calculations have also shown that the other common 5′-pyrimidine nucleotides will show similar preferences for the glycosyl and C(4′)–C(5′) bond conformations. These results are in remarkable agreement with the concept of the “rigid” nucleotide unit that has been developed from available data on mononucleotides and dinucleoside monophosphates. It is found that the conformational ‘rigidity’ in 5′-nucleotides compared with that of nucleosides is a consequence of, predominantly, the coulombic interactions between the negatively charged phosphate group and the base. The above result permits one to consider polynucleotide conformations in terms of a “rigid” C(2′)-endo or C(3′)-endo nucleotide unit with the major conformational changes being brought about by rotations about the P–O bonds linking the internucleotide phosphorus atom. IT is predicted that the anti and the gg conformations about the glycosyl and the C(4′)–C(5′) bonds would be strongly preferred in the mononucleotide components of different purine and pyrimidine coenzymes and also in the nucleotide phosphates like adenodine di- and triphosphates.     

Configuration-dependent properties of randomly coiling polynucleotide chains. II. The role of the phosphodiester linkage

The dependence of the unperturbed dimensions of randomly coiling polynucleotides on the rotations about the phosphodiester linkages of the chain has been examined in order to understand the conformational discrepancies, set forth in paper I, regarding these angles (ω′ and ω). Large values of the characteristic ratio 〈r²〉₀/nl² , which agree with the experimental behavior of the chain, are obtained only if a sizeable proportion of the polymer residues have trans ω′ values. The asymmetric torsional potential that is believed to arise from gauche effects associated with the P-O bonds has been approximated using a hard core model. The calculated characteristic ratio exhibits a strong dependence upon the magnitude of this torsional barrier (separating trans and gauche conformations) and shows agreement with experimental values for polyribonucleotides only if this energy difference is 1 kcal/mol or less.     

Theoretical conformational analysis of methylamide N-acetyl-L-arginine]

The spatial structure of methylamide N-acetyl-L-argine was studied taking into account the non-valent and electrostati interactions, the torsion energy, and the distorsion of valency angles. Calculation of the favourable conformations of the molecule was carried out with the use of all the combinations of angles phi, psi, chi1 divided by chi4 as an intital approximation. These correspond to the low energy forms of the main chain and to the minima of the torsion potentials of the side chain. Conformational possibilities of arginine and lysine were compared. The calculated stable conformation of N-acetyl-L-arginine-methylamide are compared with the geometry of arginine residues in the proteins with known structure.