A Transition-State Perspective on Y-Family DNA Polymerase η Fidelity in Comparison with X-Family DNA Polymerases λ and ß.

Deoxynucleotide misincorporation efficiencies can span a wide 104-fold range, from ∼10-2 to ∼10-6, depending principally on polymerase (pol) identity and DNA sequence context. We have addressed DNA pol fidelity mechanisms from a transition-state (TS) perspective using our "tool-kit" of dATP- and dGTP-ß,γ substrate analogues in which the pyrophosphate leaving group (p Ka4 = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning p Ka4 values from 7.8 (CF2) to 12.3 [C(CH3)2]. Here, we have used a linear free energy relationship (LFER) analysis, in the form of a Brønsted plot of log( kpol) versus p Ka4, for Y-family error-prone pol η and X-family pols λ and ß to determine the extent to which different electrostatic active site environments alter kpol values. The apparent chemical rate constant ( kpol) is the rate-determining step for the three pols. The pols each exhibit a distinct catalytic signature that differs for formation of right (A·T) and wrong (G·T) incorporations observed as changes in slopes and displacements of the Brønsted lines, in relation to a reference LFER. Common to this signature among all three pols is a split linear pattern in which the analogues containing two halogens show kpol values that are systematically lower than would be predicted from their p Ka4 values measured in aqueous solution. We discuss how metal ions and active site amino acids are responsible for causing "effective" p Ka4 values that differ for dihalo and non-dihalo substrates as well as for individual R and S stereoisomers for CHF and CHCl.

Computer simulations of the catalytic mechanism of wild-type and mutant ß-phosphoglucomutase.

ß-Phosphoglucomutase (ß-PGM) has served as an important model system for understanding biological phosphoryl transfer. This enzyme catalyzes the isomerization of ß-glucose-1-phosphate to ß-glucose-6-phosphate in a two-step process proceeding via a bisphosphate intermediate. The conventionally accepted mechanism is that both steps are concerted processes involving acid-base catalysis from a nearby aspartate (D10) side chain. This argument is supported by the observation that mutation of D10 leaves the enzyme with no detectable activity. However, computational studies have suggested that a substrate-assisted mechanism is viable for many phosphotransferases. Therefore, we carried out empirical valence bond (EVB) simulations to address the plausibility of this mechanistic alternative, including its role in the abolished catalytic activity of the D10S, D10C and D10N point mutants of ß-PGM. In addition, we considered both of these mechanisms when performing EVB calculations of the catalysis of the wild type (WT), H20A, H20Q, T16P, K76A, D170A and E169A/D170A protein variants. Our calculated activation free energies confirm that D10 is likely to serve as the general base/acid for the reaction catalyzed by the WT enzyme and all its variants, in which D10 is not chemically altered. Our calculations also suggest that D10 plays a dual role in structural organization and maintaining electrostatic balance in the active site. The correct positioning of this residue in a catalytically competent conformation is provided by a functionally important conformational change in this enzyme and by the extensive network of H-bonding interactions that appear to be exquisitely preorganized for the transition state stabilization.

DNA Polymerase λ Active Site Favors a Mutagenic Mispair between the Enol Form of Deoxyguanosine Triphosphate Substrate and the Keto Form of Thymidine Template: A Free Energy Perturbation Study.

Human DNA polymerase λ is an intermediate fidelity member of the X family, which plays a role in DNA repair. Recent X-ray diffraction structures of a ternary complex of a loop-deletion mutant of polymerase λ, a deoxyguanosine triphosphate analogue, and a gapped DNA show that guanine and thymine form a mutagenic mispair with an unexpected Watson-Crick-like geometry rather than a wobble geometry. Hence, there is an intriguing possibility that either thymine in the DNA or guanine in the deoxyguanosine triphosphate analogue may spend a substantial fraction of time in a deprotonated or enol form (both are minor species in aqueous solution) in the active site of the polymerase λ mutant. The experiments do not determine particular forms of the nucleobases that contribute to this mutagenic mispair. Thus, we investigate the thermodynamics of formation of various mispairs between guanine and thymine in the ternary complex at a neutral pH using classical molecular dynamics simulations and the free energy perturbation method. Our free energy calculations, as well as a comparison of the experimental and computed structures of mispairs, indicate that the Watson-Crick-like mispair between the enol tautomer of guanine and the keto tautomer of thymine is dominant. The wobble mispair between the keto forms of guanine and thymine and the Watson-Crick-like mispair between the keto tautomer of guanine and the enol tautomer of thymine are less prevalent, and mispairs that involve deprotonated guanine or thymine are thermodynamically unlikely. These findings are consistent with the experiment and relevant for understanding mechanisms of spontaneous mutagenesis.

Metal Ion Complexes of N,N'-Bis(2-Pyridylmethyl)-trans-1,2-Diaminocyclohexane-N,N'-Diacetic Acid, H2bpcd: Lanthanide(III)-bpcd2- Cationic Complexes.

The synthesis and characterization of N,N'-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N'-diacetic acid (H2bpcd) cationic complexes of La(III), Nd(III), and Sm(III) are reported. The Ln(III)-bpcd2- complex ions, where bpcd2- stands for N,N'-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N'-diacetate, were isolated as PF6- salts. These salts were characterized by elemental analysis, X-ray crystallography, IR, and 1H and 13C NMR spectroscopy. Binuclear [La2(bpcd)2(H2O)2]2+ crystallized from an aqueous solution in the monoclinic P21/c space group as a cocrystallate with Na2bpcd and NaPF6, nominally Na2.34[La1.22(C22H26N4O4)2(H2O)2][PF6]2·2H2O, with a = 11.3343(6) Å, b = 17.7090(9) Å, c = 15.0567(8) Å, ß = 110.632(3)°, and Z = 4 (Z' = 2). La is eight-coordinate with distorted dodecahedral coordination geometry provided by a N4O4 donor atom set. In addition to four N atoms from the bpcd2- ligand, La's coordination sphere includes O atoms from a water molecule and three acetate groups (one O atom from singly bound acetate and two O atoms from acetate groups that bridge the La centers). The 1H and 13C assignments for H2bpcd and the metal-bpcd2- complexes were made on the basis of 2D COSY and HSQC experiments, which established 1H-1H and 1H-13C correlations. The NMR spectral data were used to establish the symmetry of the cationic complexes present in aqueous solution. The data indicate that the La(III)-bpcd2- and Sm(III)-bpcd2- complexes are present in solution as a single species with C2 symmetry. The 1H NMR spectrum of [Nd(bpcd)]PF6 in D2O consists of eight considerably line-broadened, paramagnetic-shifted singlets. The ab initio quantum mechanical calculations at the PCM/MP2/SDD//HF/SDD level, which were established previously for determining isomerization energies for octahedral M(III)-bpad2- complex ions, were used to determine the relative free energies of the geometric isomers possible for eight- and nine-coordinate La(III)-bpcd2- cationic aqua complexes in aqueous solution, i.e., [La(bpcd)(H2O)2]+ and La(bpcd)(H2O)3]+.

Uniform Free-Energy Profiles of the P-O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases ß and λ.

Human X-family DNA polymerases ß (Polß) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3'-OH group of the primer DNA strand, and the subsequent formation and cleavage of P-O bonds in four Polß, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol-1 for Polß, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P-O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pKa of the 3'-OH group as a predictor of the catalytic rate of X-family DNA polymerases.

Lipid molecules can induce an opening of membrane-facing tunnels in cytochrome P450 1A2.

Cytochrome P450 1A2 (P450 1A2, CYP1A2) is a membrane-bound enzyme that oxidizes a broad range of hydrophobic substrates. The structure and dynamics of both the catalytic and trans-membrane (TM) domains of this enzyme in the membrane/water environment were investigated using a multiscale computational approach, including coarse-grained and all-atom molecular dynamics. Starting from the spontaneous self-assembly of the system containing the TM or soluble domain immersed in randomized dilauroyl phosphatidylcholine (DLPC)/water mixture into their respective membrane-bound forms, we reconstituted the membrane-bound structure of the full-length P450 1A2. This structure includes a TM helix that spans the membrane, while being connected to the catalytic domain by a short flexible loop. Furthermore, in this model, the upper part of the TM helix interacts directly with a conserved and highly hydrophobic N-terminal proline-rich segment of the catalytic domain; this segment and the FG loop are immersed in the membrane, whereas the remaining portion of the catalytic domain remains exposed to aqueous solution. The shallow membrane immersion of the catalytic domain induces a depression in the opposite intact layer of the phospholipids. This structural effect may help in stabilizing the position of the TM helix directly beneath the catalytic domain. The partial immersion of the catalytic domain also allows for the enzyme substrates to enter the active site from either aqueous solution or phospholipid environment via several solvent- and membrane-facing tunnels in the full-length P450 1A2. The calculated tunnel dynamics indicated that the opening probability of the membrane-facing tunnels is significantly enhanced when a DLPC molecule spontaneously penetrates into the membrane-facing tunnel 2d. The energetics of the lipid penetration process were assessed by the linear interaction energy (LIE) approximation, and found to be thermodynamically feasible.

Membrane-Anchored Cytochrome P450 1A2-Cytochrome b5 Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices.

Eukaryotic cytochromes P450 (P450) are membrane-bound enzymes oxidizing a broad spectrum of hydrophobic substrates, including xenobiotics. Protein-protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates catalytic activities of several P450s. To lay a structural foundation for the investigation of these effects, we constructed a model of the membrane-bound full-length human P450 1A2-cyt b5 complex. The model was assembled from several parts using a multiscale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2-cyt b5 complexes, these simulations yielded three stable binding modes (sAI, sAII, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mAI and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mAII mode was found to be unstable in the membrane environment. The mutual position of soluble domains in binding mode mAI was analogous to the sAI complex. Featuring the largest contact area, the least structural flexibility, the shortest electron transfer distance, and the highest number of interprotein salt bridges, mode mAI is the best candidate for the catalytically relevant full-length complex.

Metal Ion Complexes of N,N'-Bis(2-Pyridylmethyl)-trans-1,2-Diaminocyclohexane-N,N'-Diacetic Acid, H2bpcd: Cis/Trans Isomerization Equilibria.

The synthesis of N,N'-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N'-diacetic acid (H2bpcd) and its complexation of Ga(III) and Co(III) are reported. H2bpcd and the metal-bpcd(2-) complexes, isolated as hexafluorophosphate salts, were characterized by elemental analysis, X-ray crystallography, IR spectroscopy, and (1)H and (13)C NMR spectroscopy. [Ga(bpcd)]PF6, [Ga(C22H26N4O4)]PF6, crystallized in the orthorhombic space group Ibca, with a = 13.8975(7) Å, b = 15.0872(7) Å, c = 22.2418(10) Å, and Z = 8. Ga is coordinated in a distorted octahedral geometry provided by a N4O2 donor atom set with trans-monodentate acetate groups and cis-2-pyridylmethyl N atoms, i.e., the trans-O,O isomer. The diamagnetic [Co(bpcd)]PF6, [Co(C22H26N4O4)]PF6, also crystallized from solution in the Ibca space group as the trans-O,O isomer. The (1)H and (13)C assignments for H2bpcd and metal-bpcd(2-) complexes were made on the basis of 2D COSY and HSQC experiments, which were used to differentiate among three possible isomers, i.e., one cis (C1 symmetry) and two trans (C2 symmetry). NMR results indicate that the [Ga(bpcd)](+), [Co(bpcd)](+), and cis-O,O, cis-Npy,Npy-[Ga(bppd)](+) cations, where bppd(2-) stands for bis(2-pyridylmethyl)-1,3-diaminopropane diacetate, are present in solution as isomers with the same symmetry as observed in the solid state. The crystallographic data and the dramatic shift that occurs in the position of the cis/trans isomerization equilibria for the [Ga(bpad)](+) cations simply by increasing the number of bridging CH2 groups in the ligand's diamine backbone represent a unique opportunity to assess the accuracy of modern computational methods. The performance of several local density functionals using a pseudopotential-based SDD basis set was compared with the more rigorous HF and MP2 ab initio calculations. The SVWN5 and SV5LYP functionals provide significantly better Ga-O and Ga-N distances than the HF method or the nonlocal BLYP functional. However, to provide proper isomerization energies the pseudopotential-DFT calculations must be augmented by MP2 single-point energies and calculations of solvation free energies.

Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.

Quantum mechanical analysis of nonenzymatic nucleotidyl transfer reactions: kinetic and thermodynamic effects of ß-γ bridging groups of dNTP substrates.

Rate (k) and equilibrium (K) constants for the reaction of tetrahydrofuranol with a series of Mg(2+) complexes of methyl triphosphate analogues, CH3O-P(O2)-O-P(O2)-X-PO3(4-), X = O, CH2, CHCH3, C(CH3)2, CFCH3, CHF, CHCl, CHBr, CFCl, CF2, CCl2, and CBr2, forming phosphate diester and pyrophosphate or bisphosphonate in aqueous solution were evaluated by B3LYP/TZVP//HF/6-31G* quantum chemical calculations and Langevin dipoles and polarized continuum solvation models. The calculated log k and log K values were found to depend linearly on the experimental pKa4 of the conjugate acid of the corresponding pyrophosphate or bisphosphonate leaving group. The calculated slopes of these Brønsted linear free energy relationships were ßlg = -0.89 and ßeq = -0.93, respectively. The studied compounds also followed the linear relationship Δlog k = 0.8Δlog K, which became less steep, Δlog k = 0.6Δlog K, after the range of studied compounds was extended to include analogues that were doubly protonated on γ-phosphate, CH3O-P(O2)-O-P(O2)-X-PO3H2(2-). The scissile Pα-Olg bond length in studied methyl triphosphate analogues slightly increases with decreasing pKa of the leaving group; concomitantly, the CH3OPα(O2) moiety becomes more positive. These structural effects indicate that substituents with low pKa can facilitate both Pα-Olg bond breaking and the Pα-Onuc bond forming process, thus explaining the large negative ßlg calculated for the transition state geometry that has significantly longer Pα-Onuc distance than the Pα-Olg distance.

Metal ion complexes of N,N'-bis(2-pyridylmethyl)-1,3-diaminopropane-N,N'-diacetic acid, H2bppd.

A higher yield synthesis of N,N'-bis(2-pyridylmethyl)-1,3-diaminopropane-N,N'-diacetic acid (H2bppd) and its complexation of trivalent metal ions (Al(III), Ga(III), In(III)) and selected lanthanides (Ln(III)) are reported. H2bppd and the metal-bppd(2-) complexes, isolated as hexafluorophosphate salts, were characterized by elemental analysis, mass spectrometry, IR, and (1)H and (13)C NMR spectroscopy. [Ga(bppd)]PF6, [Ga(C19H22N4O4)]PF6, was crystallized as colorless needles by slow evaporation from anhydrous methanol; its molecular structure was solved by direct X-ray crystallography methods. The compound crystallized in the monoclinic space group P21/c, with a = 9.6134(2) Å, b = 20.2505(4) Å, c = 11.6483(3) Å, ß = 97.520(1)(o), and Z = 4. Ga is coordinated in a distorted octahedral geometry provided by a N4O2 donor atom set with cis-monodentate acetate groups and cis-2-pyridylmethyl N atoms. Quantum mechanical calculations were performed for the three possible geometric isomers of a pseudo-octahedral metal-bppd(2-) complex with five different metal ions. The results indicate, that in aqueous solution, the stability of the trans-O,O isomer is similar to that of the cis-O,O; cis-Npy,Npy isomer but is greater than that of the trans-Npy,Npy isomer. Calculations for a six-coordinate La(III)-bppd(2-) complex converge to a structure with a very large Npy-La-Npy bond angle (146.4°), a high metal charge (2.28 au), and a high solvation free energy (-79.4 kcal/mol). The most stable geometric arrangement for bppd(2-) around the larger La(III) is best described as an open nestlike structure with space available for additional ligands. IR spectroscopy was used to investigate the nature of the H2bppd-metal complexes isolated in the solid state and the binding modes of the carboxylate functionalities. The spectra indicate that fully deprotonated [M(bppd)](+) complexes as well as partially protonated complexes [M(Hbppd)Cl](+) were isolated. The (1)H and (13)C assignments for H2bppd and metal-bppd(2-) complexes were made on the basis of 2D COSY, NOESY, and (1)H-(13)C HSQC experiments, which were used to differentiate among the cis (C1 symmetry) and the two trans (C2 symmetry) isomers.

Empirical valence bond simulations of the chemical mechanism of ATP to cAMP conversion by anthrax edema factor.

The two-metal catalysis by the adenylyl cyclase domain of the anthrax edema factor toxin was simulated using the empirical valence bond (EVB) quantum mechanical/molecular mechanical approach. These calculations considered the energetics of the nucleophile deprotonation and the formation of a new P-O bond in aqueous solution and in the enzyme-substrate complex present in the crystal structure models of the reactant and product states of the reaction. Our calculations support a reaction pathway that involves metal-assisted transfer of a proton from the nucleophile to the bulk aqueous solution followed by subsequent formation of an unstable pentavalent intermediate that decomposes into cAMP and pyrophosphate (PPi). This pathway involves ligand exchange in the first solvation sphere of the catalytic metal. At 12.9 kcal/mol, the barrier for the last step of the reaction, the cleavage of the P-O bond to PPi, corresponds to the highest point on the free energy profile for this reaction pathway. However, this energy is too close to the value of 11.4 kcal/mol calculated for the barrier of the nucleophilic attack step to reach a definitive conclusion about the rate-limiting step. The calculated reaction mechanism is supported by reasonable agreement between the experimental and calculated catalytic rate constant decrease caused by the mutation of the active site lysine 346 to arginine.

Catalytic effects of mutations of distant protein residues in human DNA polymerase ß: theory and experiment.

We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase ß (Pol ß). Since the mutated residues in the point mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol ß catalytic site, we assumed that the WT and its point mutants share the same dianionic phosphorane transition-state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate. On the basis of this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Ω = (k(pol)/K(D))(mut)/(k(pol)/K(D))(WT) using free-energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences. We have performed a presteady kinetic analysis for the six mutants for comparison with wild-type Pol ß using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Ω values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Ω values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Ω values. These scaled FEP and FEP/LIE methods were also used to predict log Ω for R283A and R283L Pol ß mutants.

Intramolecular base stacking of dinucleoside monophosphate anions in aqueous solution.

Time-dependent motions of 32 deoxyribodinucleoside and ribodinucleoside monophosphate anions in aqueous solution at 310 K were monitored during 40 ns using classical molecular dynamics (MD). In all studied molecules, spontaneous stacking/unstacking transitions occurred on a time-scale of 10 ns. To facilitate the structural analysis of the sampled configurations we defined a reaction coordinate for the nucleobase stacking that considers both the angle between the planes of the two nucleobases and the distance between their mass-centers. Additionally, we proposed a physically meaningful transient point on this coordinate that separates the stacked and unstacked states. We applied this definition to calculate free energies for stacking of all pairwise combinations of adenine, thymine (uracil), cytosine and guanine moieties embedded in studied dinucleosides monophosphate anions. The stacking equilibrium constants decreased in the order 5'-AG-3' > GA ~ GG ~ AA > GT ~ TG ~ AT ~ GC ~ AC > CG ~ TA > CA ~ TC ~ TT ~ CT ~ CC. The stacked conformations of AG occurred 10 times more frequently than its unstacked conformations. On the other hand, the last five base combinations showed a greater preference for the unstacked than the stacked state. The presence of an additional 2'-OH group in the RNA-based dinucleoside monophosphates increased the fraction of stacked complexes but decreased the compactness of the stacked state. The calculated MD trajectories were also used to reveal prevailing mutual orientation of the nucleobase dipoles in the stacked state.

Evaluation of tomosynthesis elastography in a breast-mimicking phantom.

OBJECTIVE: To evaluate whether measurement of strain under static compression in tomosynthesis of a breast-mimicking phantom can be used to distinguish tumor-simulating lesions of different elasticities and to compare the results to values predicted by rheometric analysis as well as results of ultrasound elastography. MATERIALS AND METHODS: We prepared three soft breast-mimicking phantoms containing simulated tumors of different elasticities. The phantoms were imaged using a wide angle tomosynthesis system with increasing compression settings ranging from 0 N to 105 N in steps of 15 N. Strain of the inclusions was measured in two planes using a commercially available mammography workstation. The elasticity of the phantom matrix and inclusion material was determined by rheometric analysis. Ultrasound elastography of the inclusions was performed using two different ultrasound elastography algorithms. RESULTS: Strain at maximal compression was 24.4%/24.5% in plane 1/plane 2, respectively, for the soft inclusion, 19.6%/16.9% for the intermediate inclusion, and 6.0%/10.2% for the firm inclusion. The strain ratios predicted by rheometrical testing were 0.41, 0.83 and 1.26 for the soft, intermediate, and firm inclusions, respectively. The strain ratios obtained for the soft, intermediate, and firm inclusions were 0.72±0.13, 1.02±0.21 and 2.67±1.70, respectively for tomosynthesis elastography, 0.91, 1.64 and 2.07, respectively, for ultrasound tissue strain imaging, and 0.97, 2.06 and 2.37, respectively, for ultrasound real-time elastography. CONCLUSIONS: Differentiation of tumor-simulating inclusions by elasticity in a breast mimicking phantom may be possible by measuring strain in tomosynthesis. This method may be useful for assessing elasticity of breast lesions tomosynthesis of the breast.

CH···π interactions do not contribute to hydrogen transfer catalysis by glycerol dehydratase.

The role of the nonbonded CH···π interaction in the hydrogen abstraction from glycerol by the coenzyme B(12)-independent glycerol dehydratase (GDH) was examined using the QM/MM (ONIOM), MP2, and CCSD(T) methods. The studied CH···π interaction included the hydrogen atom of the -C(2)H(OH)- group of the glycerol substrate and the tyrosine-339 residue of the dehydratase. A contribution of this interaction to the stabilization of the transition state for the transfer of a hydrogen atom from the adjacent terminal C(1)H(2)(OH) group to cysteine 433 was determined by ab initio HF, MP2, and CCSD(T) calculations with the aug-cc-pvDZ basis set for the corresponding methane/benzene, methanol/phenol, and glycerol radical/phenol subsystems. The calculated CH···π distance, defined as the distance between the H atom and the center of the phenol ring, shortened from 2.62 to 2.52 Å upon going from the ground- to the transition-state of the GDH-catalyzed reaction. However, this shortening was not accompanied by the expected lowering of the CH···π interaction free energy. Instead, this interaction remained weak (about -1 kcal/mol) along the entire reaction coordinate. Additionally, the mutual orientation of the CH group and the phenol ring did not change significantly during the reaction. These results suggest that the phenol group of the tyrosine-339 does not contribute to lowering the activation barrier in the enzyme, but do not exclude the possibility that tyrosine 339 facilitates proper orientation of glycerol for the electrostatic catalysis, or inhibits side-reactions of the reactive glycerol radical intermediate.

An abridged transition state model to derive structure, dynamics, and energy components of DNA polymerase ß fidelity.

We show how a restricted reaction surface can be used to facilitate the calculation of biologically important contributions of active site geometries and dynamics to DNA polymerase fidelity. Our analysis, using human DNA polymerase beta (pol ß), is performed within the framework of an electrostatic linear free energy response (EFER) model. The structure, dynamics, and energetics of pol ß-DNA-dNTP interactions are computed between two points on the multidimensional reaction free energy surface. "Point 1" represents a ground state activation intermediate (GSA), which is obtained by deprotonating the terminal 3'OH group of the primer DNA strand. "Point 2" is the transition state (PTS) for the attack of the 3'O(-) (O(nuc)) on the P(α) atom of dNTP substrate, having the electron density of a dianionic phosphorane intermediate. Classical molecular dynamics simulations are used to compute the geometric and dynamic contributions to the formation of right and wrong O(nuc)-P chemical bonds. Matched dCTP·G and mismatched dATP·G base pairs are used to illustrate the analysis. Compared to the dCTP·G base pair, the dATP·G mismatch has fewer GSA configurations with short distances between O(nuc) and P(α) atoms and between the oxygen in the scissile P-O bond (O(lg)) and the nearest structural water. The thumb subdomain conformation of the GSA complex is more open for the mismatch, and the H-bonds in the mispair become more extended during the nucleophilic attack than in the correct pair. The electrostatic contributions of pol ß and DNA residues to catalysis of the right and wrong P-O(nuc) bond formation are 5.3 and 3.1 kcal/mol, respectively, resulting in an 80-fold contribution to fidelity. The EFER calculations illustrate the considerable importance of Arg183 and an O(lg)-proximal water molecule to pol ß fidelity.

QM/MM (ONIOM) study of glycerol binding and hydrogen abstraction by the coenzyme B12-independent dehydratase.

Glycerol binding and the radical-initiated hydrogen transfer by the coenzyme B(12)-independent glycerol dehydratase from Clostridium butyricum were investigated by using quantum mechanical/molecular mechanical (QM/MM) calculations based on the high-resolution crystal structure (PDB code: 1r9d). Our QM/MM calculations of enzyme catalysis considered the electrostatic coupling between the quantum-mechanical and molecular-mechanical subsystems and two alternative mechanisms. In addition to performing QM/MM calculations in the enzyme, we evaluated energetics along the same reaction pathway in aqueous solution modeled by the polarized dielectric and in the virtual enzyme site that included full steric component from the enzyme residues described by molecular mechanics but lacked the electrostatic contribution of these residues. In this way, we established significant enzyme catalytic effect with respect to reference reactions in both an aqueous solution and a nonpolar cavity. Structurally, four hydrogen bonds formed between glycerol and H164, S282, E435, and D447 anchor glycerol for hydrogen abstraction by thiyl radical on C433. These hydrogen-bond partners orient glycerol molecule to facilitate the formation of the transition state for hydrogen abstraction from carbon C1. This reaction then proceeds with the activation free energy of 6.3 kcal/mol and the reaction free energy of 6.1 kcal/mol. The polarization effects imposed by these hydrogen bonds represent a predominant contribution to a 7.5 kcal/mol enzyme catalytic effect. These results demonstrate the importance of electrostatic catalysis and hydrogen-bonding in enzyme-catalyzed radical reactions and advance our understanding of the catalytic mechanism of B(12)-independent glycerol dehydratases.

DNA duplex stability: the role of preorganized electrostatics.

The insertion of a DNA base moiety at the end of a DNA duplex to form a Watson-Crick or wobble pair during DNA annealing or replication is a step of fundamental biological importance. Therefore, we investigated the energetics of a formation of the terminal G x C, G x T, and G x A base pairs in DNA containing a 5'-dangling G adjacent to the base insertion point using differential scanning calorimetry and computer simulations. The energies calculated along classical molecular dynamics trajectories in aqueous solution were analyzed in the framework of linear-response approximation (LRA) to obtain relative free energies for the base insertion and their electrostatic, van der Waals, and preorganization components. Using the generic set of LRA parameters, the calculated free energies disfavored the mispair formation by 2.5 (G x C --> G x T) and 1.7 (G x C --> G x A) kcal/mol, in reasonable agreement with the experimental free energy differences of 1.8 and 1.4 kcal/mol, respectively. The calculated preorganization components of these free energies of 0.6 (G x C --> G x T) and -0.1 (G x C --> G x A) kcal/mol show that electrostatic preorganization, which is an important source of DNA replication fidelity, plays a lesser role in the mispair destabilization in the absence of DNA polymerase.