Our overall goal is to develop the computational infrastructure to enable pH to be treated the same level as temperature and pressure in molecular dynamics simulation studies of biological molecules. These tools and methods need to be robust and straightforward to employ. Our work to date in this area has had a significant impact on the field;others have implemented similar methods, and key applications demonstrate the utility of direct incorporation of pH effects into molecular simulations.
Our specific aims are directed toward the establishment of a robust methodology for explicit solvent constant pH simulations and are driven by applications to nucleic acids and to a specific protein system where we will explore pH sensing elements in the chaperon activity of a conditionally disordered protein.
Aim 1 is to develop novel and robust explicit solvent CpHMD methods for nucleic acids. Nucleic acids present special challenges to implicit solvent models because of strong electrostatic fields from the nucleic acid polyanion and the significant influences of their ion atmosphere. Explicit solvent, with explicit ions, provides an alternative to the implicit solvet models. New developments in CpHMD will extend the approach to use an explicit solvent representation and will improve the precision, accuracy and robustness of the methodology. Our developments will be benchmarked against RNA and DNA measured pKa values for key bases, predominately the bases A and C, and through explicit collaborations with experimental colleagues.
Aim 2 will apply CpHMD to explore the role of pH in mediating DNA/RNA conformation and base-base interactions. We will examine a number of specific cases, pH-mediated conformational switching in the SARS-CoV coronavirus, in the U6 intra-molecular stem loop of the splicosome, and the role of pH in the catalytic mechanism of the hairpin ribozyme. These applications address fundamental questions regarding the role of pH-driven protonation equilibrium in structure, dynamics and function in nucleic acids. Collaborations with the Al-Hashimi and Walter groups at Michigan and with Victoria D'Souza from Harvard provide both experimental benchmarks for our calculations and enable additional experiment and analysis to be informed from the calculations.
Aim 3 aims to apply CpHMD to delineate and redesign the pH sensing mechanism in the Bacterial pH stress response protein chaperon HdeA. With the Bardwell group, we have redesigned the pH response elements in HdeA to produce a constitutively active and intrinsically disordered protein at pH 7. This provides a basis for exploration of the interactions within the largely disordered, but chaperon active, protein and the interactions it makes with its substrates. Collaboration with the Bardwell and Al-Hashimi groups will combine, coarse-grained simulations with CpHMD and NMR to explore the nature of the active disordered ensemble and how its members interact with substrate molecules.
The role that pH plays in mediating biological function, including protein folding and misfolding, viral infectivity, biochemical catalysis by proteins and nucleic acids, is ubiquitious and thereby has important consequences in understanding biological processes associated with human disease. This proposed work aims to establish a robust theoretical and computational framework to enable the computational investigation of pH mediated processes of biomedical interest, from the pH sensing and chaperone mechanism of enteric Bacterial proteins to the role of pH mediated RNA dynamics in the SAR-CoV coronavirus frameshifting.