A Multidimensional B-Spline Correction for Accurate Modeling Sugar Puckering in QM/MM Simulations.

The computational efficiency of approximate quantum mechanical methods allows their use for the construction of multidimensional reaction free energy profiles. It has recently been demonstrated that quantum models based on the neglect of diatomic differential overlap (NNDO) approximation have difficulty modeling deoxyribose and ribose sugar ring puckers and thus limit their predictive value in the study of RNA and DNA systems. A method has been introduced in our previous work to improve the description of the sugar puckering conformational landscape that uses a multidimensional B-spline correction map (BMAP correction) for systems involving intrinsically coupled torsion angles. This method greatly improved the adiabatic potential energy surface profiles of DNA and RNA sugar rings relative to high-level ab initio methods even for highly problematic NDDO-based models. In the present work, a BMAP correction is developed, implemented, and tested in molecular dynamics simulations using the AM1/d-PhoT semiempirical Hamiltonian for biological phosphoryl transfer reactions. Results are presented for gas-phase adiabatic potential energy surfaces of RNA transesterification model reactions and condensed-phase QM/MM free energy surfaces for nonenzymatic and RNase A-catalyzed transesterification reactions. The results show that the BMAP correction is stable, efficient, and leads to improvement in both the potential energy and free energy profiles for the reactions studied, as compared with ab initio and experimental reference data. Exploration of the effect of the size of the quantum mechanical region indicates the best agreement with experimental reaction barriers occurs when the full CpA dinucleotide substrate is treated quantum mechanically with the sugar pucker correction.

Multiscale methods for computational RNA enzymology.

RNA catalysis is of fundamental importance to biology and yet remains ill-understood due to its complex nature. The multidimensional "problem space" of RNA catalysis includes both local and global conformational rearrangements, changes in the ion atmosphere around nucleic acids and metal ion binding, dependence on potentially correlated protonation states of key residues, and bond breaking/forming in the chemical steps of the reaction. The goal of this chapter is to summarize and apply multiscale modeling methods in an effort to target the different parts of the RNA catalysis problem space while also addressing the limitations and pitfalls of these methods. Classical molecular dynamics simulations, reference interaction site model calculations, constant pH molecular dynamics (CpHMD) simulations, Hamiltonian replica exchange molecular dynamics, and quantum mechanical/molecular mechanical simulations will be discussed in the context of the study of RNA backbone cleavage transesterification. This reaction is catalyzed by both RNA and protein enzymes, and here we examine the different mechanistic strategies taken by the hepatitis delta virus ribozyme and RNase A.

Interpretation of pH-activity profiles for acid-base catalysis from molecular simulations.

The measurement of reaction rate as a function of pH provides essential information about mechanism. These rates are sensitive to the pK(a) values of amino acids directly involved in catalysis that are often shifted by the enzyme active site environment. Experimentally observed pH-rate profiles are usually interpreted using simple kinetic models that allow estimation of "apparent pK(a)" values of presumed general acid and base catalysts. One of the underlying assumptions in these models is that the protonation states are uncorrelated. In this work, we introduce the use of constant pH molecular dynamics simulations in explicit solvent (CpHMD) with replica exchange in the pH-dimension (pH-REMD) as a tool to aid in the interpretation of pH-activity data of enzymes and to test the validity of different kinetic models. We apply the methods to RNase A, a prototype acid-base catalyst, to predict the macroscopic and microscopic pK(a) values, as well as the shape of the pH-rate profile. Results for apo and cCMP-bound RNase A agree well with available experimental data and suggest that deprotonation of the general acid and protonation of the general base are not strongly coupled in transphosphorylation and hydrolysis steps. Stronger coupling, however, is predicted for the Lys41 and His119 protonation states in apo RNase A, leading to the requirement for a microscopic kinetic model. This type of analysis may be important for other catalytic systems where the active forms of the implicated general acid and base are oppositely charged and more highly correlated. These results suggest a new way for CpHMD/pH-REMD simulations to bridge the gap with experiments to provide a molecular-level interpretation of pH-activity data in studies of enzyme mechanisms.

Experimental and computational analysis of the transition state for ribonuclease A-catalyzed RNA 2'-O-transphosphorylation.

Enzymes function by stabilizing reaction transition states; therefore, comparison of the transition states of enzymatic and nonenzymatic model reactions can provide insight into biological catalysis. Catalysis of RNA 2'-O-transphosphorylation by ribonuclease A is proposed to involve electrostatic stabilization and acid/base catalysis, although the structure of the rate-limiting transition state is uncertain. Here, we describe coordinated kinetic isotope effect (KIE) analyses, molecular dynamics simulations, and quantum mechanical calculations to model the transition state and mechanism of RNase A. Comparison of the (18)O KIEs on the 2'O nucleophile, 5'O leaving group, and nonbridging phosphoryl oxygens for RNase A to values observed for hydronium- or hydroxide-catalyzed reactions indicate a late anionic transition state. Molecular dynamics simulations using an anionic phosphorane transition state mimic suggest that H-bonding by protonated His12 and Lys41 stabilizes the transition state by neutralizing the negative charge on the nonbridging phosphoryl oxygens. Quantum mechanical calculations consistent with the experimental KIEs indicate that expulsion of the 5'O remains an integral feature of the rate-limiting step both on and off the enzyme. Electrostatic interactions with positively charged amino acid site chains (His12/Lys41), together with proton transfer from His119, render departure of the 5'O less advanced compared with the solution reaction and stabilize charge buildup in the transition state. The ability to obtain a chemically detailed description of 2'-O-transphosphorylation transition states provides an opportunity to advance our understanding of biological catalysis significantly by determining how the catalytic modes and active site environments of phosphoryl transferases influence transition state structure.

A variational linear-scaling framework to build practical, efficient next-generation orbital-based quantum force fields.

We introduce a new hybrid molecular orbital/density-functional modified divide-and-conquer (mDC) approach that allows the linear-scaling calculation of very large quantum systems. The method provides a powerful framework from which linear-scaling force fields for molecular simulations can be developed. The method is variational in the energy, and has simple, analytic gradients and essentially no break-even point with respect to the corresponding full electronic structure calculation. Furthermore, the new approach allows intermolecular forces to be properly balanced such that non-bonded interactions can be treated, in some cases, to much higher accuracy than the full calculation. The approach is illustrated using the second-order self-consistent charge density-functional tight-binding model (DFTB2). Using this model as a base Hamiltonian, the new mDC approach is applied to a series of water systems, where results show that geometries and interaction energies between water molecules are greatly improved relative to full DFTB2. In order to achieve substantial improvement in the accuracy of intermolecular binding energies and hydrogen bonded cluster geometries, it was necessary to extend the DFTB2 model to higher-order atom-centered multipoles for the second-order self-consistent intermolecular electrostatic term. Using generalized, linear-scaling electrostatic methods, timings demonstrate that the method is able to calculate a water system of 3000 atoms in less than half of a second, and systems of up to one million atoms in only a few minutes using a conventional desktop workstation.

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA.

Endogenous 5-methylcytosine ((Me)C) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational 'hotspots' for smoking induced lung cancer. (Me)C enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5'-CCCGGCACCC GC[(15)N(3),(13)C(1)-G]TCCGCG-3', + strand) were prepared containing [(15)N(3), (13)C(1)]-guanine opposite unsubstituted cytosine, (Me)C, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2'-deoxynucleosides, N(2)-BPDE-dG adducts formed at the [(15)N(3), (13)C(1)]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N(2)-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N(2) position of guanine.

DNA oligomers containing site-specific and stereospecific exocyclic deoxyadenosine adducts of 1,2,3,4-diepoxybutane: synthesis, characterization, and effects on DNA structure.

1,2,3,4-Diepoxybutane (DEB) is a carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is considered the ultimate carcinogenic species of BD because of its potent genotoxicity and mutagenicity attributed to its ability to form DNA-DNA cross-links and exocyclic nucleoside adducts. Mutagenesis studies suggest that DEB adducts formed at adenine bases may be critically important, as it induces large numbers of A → T transversions. We have recently identified three types of exocyclic DEB-dA lesions: N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA), 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-γ-HMHP-dA), and 1,N6-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N6-α-HMHP-dA) [Seneviratne, U., et al. (2010) Chem. Res. Toxicol. 23, 118-133]. In the work presented here, a postsynthetic methodology for preparing DNA oligomers containing stereospecific and site-specific N6,N6-DHB-dA and 1,N6-γ-HMHP-dA adducts was developed. DNA oligomers containing site-specific 6-chloropurine were coupled with optically pure 1-amino-2-hydroxy-3,4-epoxybutanes to generate oligomers containing N6-(2-hydroxy-3,4-epoxybut-1-yl)-2'-deoxyadenosine adducts, followed by their spontaneous cyclization to 1,N6-γ-HMHP-dA lesions. N6,N6-DHB-dA containing strands were prepared analogously by coupling 6-chloropurine containing DNA with (3S,4S)- or (3R,4R)-pyrrolidine-3,4-diols. Oligodeoxynucleotide structures were confirmed by ESI-MS, exonuclease ladder sequencing, and HPLC-MS/MS of enzymatic digests. UV melting and CD spectroscopy studies of DNA duplexes containing N6,N6-DHB-dA and 1,N6-γ-HMHP-dA revealed that both lesions lower the thermodynamic stability of DNA. Interestingly, structurally modified DNA duplexes were more thermodynamically stable when an adenine residue was placed opposite 1,N6-γ-HMHP-dA instead of thymine, suggesting that these adducts may preferentially pair with dA.