pH-regulation of RNA-mediated biological activity is an emerging discovery. Traditionally, the analysis of static structures solved near physiological pH is used to understand the structure-function relationship of such pH-regulated activity. While this approach may be appropriate for proteins, RNA have rugged energy landscapes, so conformational dynamics and other near-native structures cannot be ignored. One example is the U6 internal stem-loop (ISL) of the spliceosome, which undergoes a pH-dependent base flipping conformational change. Extracting such structural information is experimentally non-trivial, which drives the need to develop better tools that can describe the full extent of pH-dependent structural ensembles of RNA. In this work, we present an application of WExplore, a weighted ensemble sampling method enhanced with a hierarchical weight-balancing algorithm that has been implemented with GPU-accelerated CHARMM. We surveyed a range of protonated RNA structures that are implicated in pH-dependent biological activities, such as ribozymes, the spliceosome and other RNA pseudoknot structures. Using GPU-accelerated WExplore sampling, we identified other conformations under high and low pH conditions, and when combined with constant pH MD (CPHMD) simulations, we show how the RNA conformation modulates the microscopic pKa of each state. Interestingly, most of our results using �ground state� crystallographic or �lowest energy� NMR structures produced pKa values that were inconsistent with experimental pKa. However, when the structural data obtained from WExplore sampling was integrated in our CPHMD simulations, we were able to reproduce experimentally observed pKa values, which suggests that using a pH-dependent structural ensemble that encompasses low population minor states is a necessary condition for a fundamental understanding of protonated RNA structures. In conclusion, our work provides the first comprehensive validation that protonated RNA structures have multiple physiologically-relevant conformations, which may not be fully captured by experimental characterization.

Protonated RNA structures are not static: WExploring the pH-dependent ensemble of RNA

248th National Meeting (2014)

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