RNA-ligand interactions: simulation and experiment

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DESCRIPTION (provided by applicant): This project represents collaboration between established computational and experimental labs to combine biophysical methods to produce, assess, and validate the next generation of biomolecular simulation methods to study RNA molecules. In the past decade our knowledge of the widely varied functional roles of RNA molecules has exploded; RNA molecules can be regulatory or catalytic, can act as sensors, can both up- and down- regulate gene expression, and have great potential as targets for control by exogenous ligands in all phyla. The structures of RNA are modular and contain a mix of structural elements including short duplex regions, hairpin loops, internal bulges, four-way junctions, and receptor sites. These structural elements, along with their conformational changes and dynamics, are the keys to RNA function. However, these functionally important structural, dynamic, and energetic properties cannot yet be reliably predicted or fully experimentally understood. To characterize RNA structures, their structural transitions, and their local and global dynamics, a synergy of simulation and experiment is necessary. The goal of this project is to use theoretical and experimental biophysical methods to more fully develop biomolecular simulation tools to accurately describe RNA molecules, especially how they interact with small molecules, ions, and proteins, using experimental data as a benchmark to assess, validate and improve the models. Aim 1 is built around the Varkud satellite ribozyme Stem loop V RNA (SL5). This small RNA hairpin has a flexible loop that undergoes a conformational change when it binds to ions, and acts as a model system to test electrostatics, sampling, structural accuracy, and the ability of the simulations to model the subtle influences of ions on the structure and dynamics. New molecular dynamics simulations based on the latest AMBER nucleic acid force fields (ff99+parmbsc0) will assess the force field parameters and apply novel biased enhanced sampling methods to more richly sample the configurational space of the RNA loop. At the same time, NMR will further refine the solution structure, and 13C NMR relaxation experiments will measure dynamics of SL5 in the presence of different ions. Aim 2 expands the RNA model systems to the GTPase center (GAC) of the ribosome, to model ion binding, electrostatics, and antibiotic binding. NMR is used for structure and dynamics determination, 2-aminopurine fluorescence reports on folding of the GAC, and MD begins with crystal structures of the 58 nucleotide GAC. These two aims are synergistic: primary NMR data are back- calculated from the MD simulations to assess the accuracy and precision of the computational methods through direct comparison to the measured NMR data. Aim 3 takes the outcome of the comparative study to identify deficiencies and implement improvements. Force field modifications and conformational sampling will be facilitated through Hamiltonian replica exchange molecular dynamics and finally applied to the tertiary folding of the GAC RNA.