Molecular dynamics (MD) simulation, an established method for investigating the internal motions of biomolecules, is applied to thrombin protein, a critical blood coagulation cascade protease with complex, not yet fully understood regulatory mechanisms. Accelerated MD (AMD) is employed to achieve enhanced conformational sampling of more biologically relevant timescales, and nuclear magnetic resonance (NMR) experiments are used to validate and tune AMD parameters. In chapter 1, the thrombin system and its interaction with cofactor thrombomodulin (TM) is introduced. The potential contribution of dynamics to thrombin allostery is discussed, and methods for MD and AMD simulations are described. In chapter 2, the combined use of NMR and AMD to examine the dynamics of thrombin is detailed. AMD generated ensembles are shown to recapitulate NMR residual dipolar couplings (RDCs), observables that report on ms timescale dynamics. The resulting picture of thrombin depicts a stable core surrounded by highly dynamic surface loops. In chapter 3, a computational study comparing isolated thrombin with TM bound forms is reported. Community network analysis identifies two allosteric pathways from the TM binding exosite to the thrombin active site. The presence of the fourth EGF-like domain of TM, known to be essential for thrombin regulation, is shown to establish and strengthen these allosteric pathways. This essential domain is also observed to elicit enhanced dynamics and cross-correlated motion in the distal active site loops. In chapter 4, these analyses are extended to compare apo thrombin with active site inhibitor bound thrombin. Allosteric pathways between the active and TM binding exosite site are again observed and are altered by the presence of inhibitor. Residual local frustration analysis reveals a minimally frustrated thrombin core surrounded by highly frustrated surface loops. The highly frustrated contacts regions show significant overlap with regions undergoing slow timescale dynamics. In chapter 5, overarching implications for a dynamical mechanism to thrombin:TM allostery are discussed. The collective observations of thrombin active site surface loops undergoing concerted, long timescale dynamics in response to TM binding at exosite 1 strongly suggest a dynamic, allosteric mechanism in thrombin regulation

Thrombin is the central protease in the cascade of blood coagulation proteases. The structure of thrombin consists of a double β-barrel core surrounded by connecting loops and helices. Compared to chymotrypsin, thrombin has more extended loops that are thought to have arisen from insertions in the serine protease that evolved to impart greater specificity. Previous experiments showed thermodynamic coupling between ligand binding at the active site and distal exosites. We present a combined approach of molecular dynamics (MD), accelerated molecular dynamics (AMD), and analysis of the residual local frustration of apo-thrombin and active-site-bound (PPACK-thrombin). Community analysis of the MD ensembles identified changes upon active site occupation in groups of residues linked through correlated motions and physical contacts. AMD simulations, calibrated on measured residual dipolar couplings, reveal that upon active site ligation, correlated loop motions are quenched, but new ones connecting the active site with distal sites where allosteric regulators bind emerge. Residual local frustration analysis reveals a striking correlation between frustrated contacts and regions undergoing slow time scale dynamics. The results elucidate a motional network that probably evolved through retention of frustrated contacts to provide facile conversion between ensembles of states.

Maintaining safe operating conditions is a key challenge for high-performance lithium-ion battery applications. The lithium-plating reaction remains a risk during charging, but limited studies consider the highly variable charging conditions possible in commercial cells. Here we combine pseudo-2D electrochemical modeling with data visualization methods to reveal important relationships between the measurable cell voltage and difficult-to-predict Li-plating onset criteria. An extensively validated model is used to compute Li plating for thousands of multistep charging conditions spanning diverse rates, temperatures, states-of-charge, and cell aging. We observe an empirical cell operating voltage limit below which plating does not occur across all conditions, and this limit varies with the battery state-of-charge and aging. A model sensitivity analysis also indicates that, when comparing two charging voltage profiles, the capacity difference at 4.0 V correlates well with the difference in the plating onset capacity. These results encourage simple strategies for Li-plating prevention that are complementary to existing battery controls.