Integration of NMR and SAXS with Atomistic Simulations for Characterizing the Structure and Dynamics of Multi-Domain Proteins

In the seven decades since the first atomic-level structures of biomolecules were determined, the development and application of novel research methods has led to an advanced understanding of biological functions at the molecular level. In addition to experimental methods, key advances have been spurred by computer simulations, which provide an in silico representation of accumulated prior knowledge of biomolecular structure and dynamics. These models can be used both (i) as a complement to experimental results, filling in the gaps where experimental information is not accessible, and (ii) as complete representations, directing future research. Critically, the validity of either application depends on the accuracy of the models used. In this work, I aspired to combine computational and experimental methods to characterize the structure and dynamics of the flexibly linked two-domain protein MoCVNH3. In Chapter 1 I describe my motivation, and the suspected simulation artifacts observed in our preliminary simulations, which led me to investigate how accurately simulation models represent salt bridge interactions. Chapter 2 details my comparison of current models (“force fields”), for which significant variation but consistent overstabilization of salt bridges was discovered. This work motivated the development of a new force field, AMBER ff15ipq, which corrects, to some degree, the overstabilization and introduces extensive improvements, described in Chapter 3. Finally, in Chapter 4, I applied this new force field in simulations of MoCVNH3, for which I collected extensive experimental data leading to the determination of a structural ensemble. I validated the simulations against the experimental data set, and identified further directions for improvement. Overall, the work presented here demonstrates the power of integrating experimental and computational methods