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Abstract Background Bone metastasis (BM) is the most common site of disease in metastatic breast cancer (MBC) patients. BM impacts health-related quality of life (HRQoL). We tested prospectively the psychometric properties of the Bone Metastasis Quality of Life (BOMET-QoL-10) measure on MBC patients with BM. Methods Patients completed the BOMET-QoL-10 questionnaire, the Visual Analogue Scale (VAS) for pain, and a self-perceived health status item at baseline and at follow-up visits. We performed psychometric tests and calculated the effect size of specific BM treatment on patients´ HRQoL. Results Almost 70% of the 172 patients reported symptoms, 23.3% experienced irruptive pain, and over half were receiving chemotherapy. BOMET-QoL-10 proved to be a quick assessment tool performing well in readability and completion time (about 10 min) with 0–1.2% of missing/invalid data. Although BOMET-QoL-10 scores remained fairly stable during study visits, differences were observed for patient subgroups (e.g., with or without skeletal-related events or adverse effects). Scores were significantly correlated with physician-reported patient status, patient-reported pain, symptoms, and perceived health status. BOMET-QoL-10 scores also varied prospectively according to changes in pain intensity. Conclusions BOMET-QoL-10 performed well as a brief, easy-to-administer, useful, and sensitive HRQoL measure for potential use for clinical practice with MBC patients. Trial registration NCT03847220. Retrospectively registered on clinicaltrials.gov (February the 20th 2019).

Abstract Hydrothermal convection in porous geothermal reservoir systems can be seen as a double-edged sword. On the one hand, regions of upflow in convective systems can increase the geothermal energy potential of the reservoir; on the other hand, convection introduces uncertainty, because it can be difficult to locate these regions of upflow. Several predictive criteria, such as the Rayleigh number, exist to estimate whether convection might occur under certain conditions. As such, it is of interest which factors influence locations of upwelling regions and how these factors can be determined. We use the thermodynamic measure entropy production to describe the influence of spatially heterogeneous permeability on a hydrothermal convection pattern in a 2D model of a hot sedimentary aquifer system in the Perth Basin, Western Australia. To this end, we set up a Monte Carlo study with multiple ensembles. Each ensemble contains several hundred realizations of spatially heterogeneous permeability. The ensembles only differ in the horizontal spatial continuity (i.e., correlation length) of permeability. The entropy production of the simulated ensembles shows that the convection patterns in our models drastically change with the introduction and increase of a finite, lateral correlation length in permeability. An initial decrease of the average entropy production number with increasing lateral correlation length shows that fewer ensemble members show convection. When neglecting the purely conductive ensembles in our analysis, no significant change in the number of convection cells is seen for lateral correlation lengths larger than 2000 m. The result suggests that the strength of convective heat transfer is not sensitive to changes in lateral correlation length beyond a specific factor. It does, however, change strongly compared to simulations with a homogeneous permeability field. As such, while the uncertainty in spatial continuity of permeability may not strongly influence the convective heat transfer, our findings show that it is important to consider spatial heterogeneity and continuity of permeability when simulating convective heat transfer in an aquifer.

The quantification and analysis of uncertainties is important in all cases where maps and models of uncertain properties are the basis for further decisions. Once these uncertainties are identified, the logical next step is to determine how they can be reduced. Information theory provides a framework for the analysis of spatial uncertainties when different subregions are considered as random variables. In the work presented here, joint entropy, conditional entropy, and mutual information are applied for a detailed analysis of spatial uncertainty correlations. The aim is to determine (i) which areas in a spatial analysis share information, and (ii) where, and by how much, additional information would reduce uncertainties. As an illustration, a typical geological example is evaluated: the case of a subsurface layer with uncertain depth, shape and thickness. Mutual information and multivariate conditional entropies are determined based on multiple simulated model realisations. Even for this simple case, the measures not only provide a clear picture of uncertainties and their correlations but also give detailed insights into the potential reduction of uncertainties at each position, given additional information at a different location. The methods are directly applicable to other types of spatial uncertainty evaluations, especially where multiple realisations of a model simulation are analysed. In summary, the application of information theoretic measures opens up the path to a better understanding of spatial uncertainties, and their relationship to information and prior knowledge, for cases where uncertain property distributions are spatially analysed and visualised in maps and models.

The article presents a case study of a 56-year-old male patient who has a medical history of being a chain-smoker and who suffered from osteoarthritis. It is mentioned that the patient has acquired an acute renal failure and has undergone testing and treatment of Pemetrexed disodium. An investigation has revealed that kidney patients must avoid the intake of non-steroidal anti-inflammatory drugs.

Deoxynucleotide misincorporation efficiencies can span a wide 10 4 -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-β,γ substrate analogues in which the pyrophosphate leaving group (p K a4 = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning p K a4 values from 7.8 (CF 2 ) to 12.3 [C(CH 3 ) 2 ]. Here, we have used a linear free energy relationship (LFER) analysis, in the form of a Brønsted plot of log( k pol ) versus p K a4 , for Y-family error-prone pol η and X-family pols λ and β to determine the extent to which different electrostatic active site environments alter k pol values. The apparent chemical rate constant ( k pol ) 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 k pol values that are systematically lower than would be predicted from their p K a4 values measured in aqueous solution. We discuss how metal ions and active site amino acids are responsible for causing "effective" p K a4 values that differ for dihalo and non-dihalo substrates as well as for individual R and S stereoisomers for CHF and CHCl.

β-Phosphoglucomutase (β-PGM) has served as an important model system for understanding biological phosphoryl transfer. This enzyme catalyzes the isomerization of β-glucose-1-phosphate to β-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 β-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.

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.

The synthesis and characterization of N,N'-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N'-diacetic acid (H 2 bpcd) cationic complexes of La(III), Nd(III), and Sm(III) are reported. The Ln(III)-bpcd 2- complex ions, where bpcd 2- stands for N,N'-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N'-diacetate, were isolated as PF 6 - salts. These salts were characterized by elemental analysis, X-ray crystallography, IR, and 1 H and 13 C NMR spectroscopy. Binuclear [La 2 (bpcd) 2 (H 2 O) 2 ] 2+ crystallized from an aqueous solution in the monoclinic P2 1 /c space group as a cocrystallate with Na 2 bpcd and NaPF 6 , nominally Na 2.34 [La 1.22 (C 22 H 26 N 4 O 4 ) 2 (H 2 O) 2 ][PF 6 ] 2 ·2H 2 O, with a = 11.3343(6) Å, b = 17.7090(9) Å, c = 15.0567(8) Å, β = 110.632(3)°, and Z = 4 (Z' = 2). La is eight-coordinate with distorted dodecahedral coordination geometry provided by a N 4 O 4 donor atom set. In addition to four N atoms from the bpcd 2- 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 1 H and 13 C assignments for H 2 bpcd and the metal-bpcd 2- complexes were made on the basis of 2D COSY and HSQC experiments, which established 1 H- 1 H and 1 H- 13 C 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)-bpcd 2- and Sm(III)-bpcd 2- complexes are present in solution as a single species with C 2 symmetry. The 1 H NMR spectrum of [Nd(bpcd)]PF 6 in D 2 O 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)-bpad 2- complex ions, were used to determine the relative free energies of the geometric isomers possible for eight- and nine-coordinate La(III)-bpcd 2- cationic aqua complexes in aqueous solution, i.e., [La(bpcd)(H 2 O) 2 ] + and La(bpcd)(H 2 O) 3 ] + .

Human X-family DNA polymerases β (Polβ) 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 Polβ, 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 Polβ, 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 pK a of the 3'-OH group as a predictor of the catalytic rate of X-family DNA polymerases.

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.

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.

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.

Breast cancer is a burden for western societies, and an increasing one in emerging economies, because of its high incidence and enormous psychological, social, sanitary and economic costs. However, breast cancer is a preventable disease in a significant proportion. Recent developments in the armamentarium of effective drugs for breast cancer prevention (namely exemestane and anastrozole), the new recommendation from the National Institute for Health and Care Excellence to use preventative drugs in women at high risk as well as updated Guidelines from the US Preventive Services Task Force and the American Society of Clinical Oncology should give renewed momentum to the pharmacological prevention of breast cancer. In this article we review recent major developments in the field and examine their ongoing repercussion for breast cancer prevention. As a practical example, the potential impact of preventive measures in Spain is evaluated and a course of practical actions is delineated.

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.

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 βlg = -0.89 and βeq = -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 βlg calculated for the transition state geometry that has significantly longer Pα-Onuc distance than the Pα-Olg distance.

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, β = 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.

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.

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 β (Pol β). Since the mutated residues in the point mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol β 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 β 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 β mutants.

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.