Applying and improving the amber RNA force field

Thesis (Ph. D.)--University of Rochester. Department of Physics and Astronomy, 2016.RNA was believed to act as an information carrier between genes (DNA) and proteins in cells. However, RNA is now known to play a central role in many cellular functions. RNA can act as reaction catalyst, assist in peptide bond formation and regulate gene expression. In addition, RNA is also an important drug target. Molecular mechanics and molecular dynamics calculations can provide significant insight about the behavior of RNA in solution. The aim of my work is to model RNA dynamics and conformational change, refine and understand experimental results, test the force fields on small RNA molecules and identify problems and perform revisions of force field parameters. In the first work, umbrella sampling was used to model conformational preference of tandem GA base pairs. A modification to the standard Amber ff10 force field, which allowed the amino group of guanine to leave the plane of the base [J. Chem. Theory Comput., 2009, 5, 2088-2100] and form out-of-plane hydrogen bonds with a cross-strand cytosine or uracil, was needed. Previous solution structures showed that these tandem GA pairs adopt either imino (cis Watson-Crick / Watson-Crick A-G) or sheared (trans Hoogsteen / sugar edge A-G) conformations depending on the sequence and orientation of the adjacent closing base pairs. The free energy calculations allow direct comparison between simulation and experiment, and provide a benchmark for force fields. The results showed the importance of broadening the set of RNA molecules used to test force field performance. Then in the second work in collaboration with the Wedekind lab, molecular dynamic simulations were performed using the Amber force field to gain insight into the dynamics of the class II preQ1 riboswitch [Nat. Chem. Biol. 2013, 9, 353-355]. Four simulations were run each with and without the ligand present. In the presence of ligand, all four of the simulations demonstrated rearranged base pairs at the 3´ end, consistent with expected base pairing from comparative sequence analysis [RNA 2008, 14, 685-695]. In the absence of ligand, three of the simulations demonstrated similar changes in base pairing at the ligand binding site. In the final work, a new method is described for improving the backbone dihedral parameters of the Amber RNA force field by fitting to energies from quantum chemistry calculations using linear regression. To validate the new parameter sets, umbrella sampling was used to determine potentials of mean force (PMF) along the dihedral angles in explicit solvent, and then compared to the population of conformations observed in the protein data bank. Molecular dynamics simulations were also performed on a set of hairpin loops, duplexes and tetramers. Results from both umbrella sampling and molecular dynamics show improved performance as compared to the standard Amber force field

CHARMM Drude Polarizable Force Field for Glycosidic Linkages Involving Pyranoses and Furanoses

We present an extension of the CHARMM Drude polarizable force field to enable modeling of polysaccharides containing pyranose and furanose monosaccharides. The new force field parameters encompass 1↔2, 1→3, 1→4, and 1→6 pyranose–furanose linkages, 2→1 and 2→6 furanose–furanose linkages, 2→2, 2→3, and 2→4 furanose–pyranose, and 1↔1, 1→2, 1→3, 1→4, and 1→6 pyranose–pyranose linkages. For the glycosidic linkages, both simple model compounds and the full disaccharides with methylation at the reducing end were used for parameter optimization. The model compounds were chosen to be monomers or glycosidic-linked dimers of tetrahydropyran (THP) and tetrahydrofuran (THF). Target data for optimization included one- and two-dimensional potential energy scans of ω and the Φ/Ψ glycosidic dihedral angles in the model compounds and full disaccharides computed by quantum mechanical (QM) RIMP2/cc-pVQZ single point energies on MP2/6-31G­(d) optimized structures. Also included in the target data are extensive sets of QM gas phase monohydrate water–saccharide interactions, dipole moments, and molecular polarizabilities for both model compounds and full disaccharides. The resulting polarizable model is shown to be in good agreement with a range of QM data, offering a significant improvement over the additive CHARMM36 carbohydrate force field, as well as experimental data including crystal structures and conformational properties of disaccharides and a trisaccharide in aqueous solution

Drude polarizable force field parametrization of Carboxylate and N-acetyl Amine Carbohydrate derivatives

In this work, we report the development of Drude polarizable force-field parameters for the carboxylate and N-acetyl amine derivatives, extending the functionality of existing Drude polarizable carbohydrate force field. The force field parameters have been developed in a hierarchical manner, reproducing the quantum mechanical (QM) gas-phase properties of small model compounds representing the key functional group in the carbohydrate derivatives, including optimization of the electrostatic and bonded parameters. The optimized parameters were then used to generate the models for carboxylate and N-acetyl amine carbohydrate derivatives. The transferred parameters were further tested and optimized to reproduce crystal geometries and J-coupling data from NMR experiments. The parameter development resulted in the incorporation of D-glucuronate, L-iduronate, N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-galactosamine (GalNAc) sugars into the Drude polarizable force field. The parameters developed in this study were then applied to study the conformational properties of glycosaminoglycan polymer hyaluronan, composed of D-glucuronate and N-acetyl-D-glucosamine, in aqueous solution. Upon comparing the results from the additive and polarizable simulations it was found that the inclusion of polarization improved the description of the electrostatic interactions observed in hyaluronan resulting in enhanced conformational flexibility. The developed Drude polarizable force field parameters in conjunction with the remainder of the Drude polarizable force field parameters can be used for the future studies involving carbohydrates and their conjugates in complex, heterogeneous systems

Modified Amber Force Field Correctly Models the Conformational Preference for Tandem GA pairs in RNA

Molecular mechanics with all-atom models was used to understand the conformational preference of tandem guanine-adenine (GA) noncanonical pairs in RNA. These tandem GA pairs play important roles in determining stability, flexibility, and structural dynamics of RNA tertiary structures. Previous solution structures showed that these tandem GA pairs adopt either imino (cis Watson–Crick/Watson–Crick A-G) or sheared (trans Hoogsteen/sugar edge A-G) conformations depending on the sequence and orientation of the adjacent closing base pairs. The solution structures (GCGGACGC)₂ [<i>Biochemistry</i>, <b>1996</b>, <i>35</i>, 9677–9689] and (GCGGAUGC)₂ [<i>Biochemistry</i>, <b>2007</b>, <i>46</i>, 1511–1522] demonstrate imino and sheared conformations for the two central GA pairs, respectively. These systems were studied using molecular dynamics and free energy change calculations for conformational changes, using umbrella sampling. For the structures to maintain their native conformations during molecular dynamics simulations, a modification to the standard Amber ff10 force field was required, which allowed the amino group of guanine to leave the plane of the base [<i>J. Chem. Theory Comput</i>., <b>2009</b>, <i>5</i>, 2088–2100] and form out-of-plane hydrogen bonds with a cross-strand cytosine or uracil. The requirement for this modification suggests the importance of out-of-plane hydrogen bonds in stabilizing the native structures. Free energy change calculations for each sequence demonstrated the correct conformational preference when the force field modification was used, but the extent of the preference is underestimated

Revised RNA Dihedral Parameters for the Amber Force Field Improve RNA Molecular Dynamics

The backbone dihedral parameters of the Amber RNA force field were improved by fitting using multiple linear regression to potential energies determined by quantum chemistry calculations. Five backbone and four glycosidic dihedral parameters were fit simultaneously to reproduce the potential energies determined by a high-level density functional theory calculation (B97D3 functional with the AUG-CC-PVTZ basis set). Umbrella sampling was used to determine conformational free energies along the dihedral angles, and these better agree with the population of conformations observed in the protein data bank for the new parameters than for the conventional parameters. Molecular dynamics simulations performed on a set of hairpin loops, duplexes and tetramers with the new parameter set show improved modeling for the structures of tetramers CCCC, CAAU, and GACC, and an RNA internal loop of noncanonical pairs, as compared to the conventional parameters. For the tetramers, the new parameters largely avoid the incorrect intercalated structures that dominate the conformational samples from the conventional parameters. For the internal loop, the major conformation solved by NMR is stable with the new parameters, but not with the conventional parameters. The new force field performs similarly to the conventional parameters for the UUCG and GCAA hairpin loops and the [U­(UA)₆A]₂ duplex

Revised RNA Dihedral Parameters for the Amber Force Field Improve RNA Molecular Dynamics

The backbone dihedral parameters of the Amber RNA force field were improved by fitting using multiple linear regression to potential energies determined by quantum chemistry calculations. Five backbone and four glycosidic dihedral parameters were fit simultaneously to reproduce the potential energies determined by a high-level density functional theory calculation (B97D3 functional with the AUG-CC-PVTZ basis set). Umbrella sampling was used to determine conformational free energies along the dihedral angles, and these better agree with the population of conformations observed in the protein data bank for the new parameters than for the conventional parameters. Molecular dynamics simulations performed on a set of hairpin loops, duplexes and tetramers with the new parameter set show improved modeling for the structures of tetramers CCCC, CAAU, and GACC, and an RNA internal loop of noncanonical pairs, as compared to the conventional parameters. For the tetramers, the new parameters largely avoid the incorrect intercalated structures that dominate the conformational samples from the conventional parameters. For the internal loop, the major conformation solved by NMR is stable with the new parameters, but not with the conventional parameters. The new force field performs similarly to the conventional parameters for the UUCG and GCAA hairpin loops and the [U­(UA)₆A]₂ duplex

Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.

PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs

Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.

PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs