Due to high sensitivity of biomolecular systems to the electrostatic environments, coupled treatment of conformational and protonation equilibria is required for an accurate characterization of true ensemble of a given system. The research presented in this dissertation examines the effects of conformational and protonation equilibria of varying extent on diverse aspects of computational biomolecular modeling, as introduced in Chapter 1. The effects of protonation and stereoisomerism of two histidines on virtual screening against the M. tuberculosis enzyme RmlC are presented in Chapter 2. In Chapter 3, conformational flexibility of three M. tuberculosis prenyl synthases is probed using molecular dynamics simulations, with implications for computer-aided drug discovery effort for the new generation antibacterial and antivirulence therapeutics. Chapters 4 and 5 consider the conformational and protonation equilibria simultaneously by utilizing constant pH molecular dynamics, in which fluctuations in both conformation and protonation state are possible. In Chapter 4, a computational protocol utilizing constant pH molecular dynamics to compute pH- dependent binding free energies is presented. The methodology is further applied to protein-ligand complexes in Chapter 5, where the thermodynamic linkage between protonation equilibria, conformational dynamics, and inhibitor binding is illustrated

With the rise in antibiotic resistance, there is interest in discovering new drugs active against new targets. Here, we investigate the dynamic structures of three isoprenoid synthases from Mycobacterium tuberculosis using molecular dynamics (MD) methods with a view to discovering new drug leads. Two of the enzymes, cis-farnesyl diphosphate synthase (cis-FPPS) and cis-decaprenyl diphosphate synthase (cis-DPPS), are involved in bacterial cell wall biosynthesis, while the third, tuberculosinyl adenosine synthase (Rv3378c), is involved in virulence factor formation. The MD results for these three enzymes were then compared with previous results on undecaprenyl diphosphate synthase (UPPS) by means of active site volume fluctuation and principal component analyses. In addition, an analysis of the binding of prenyl diphosphates to cis-FPPS, cis-DPPS, and UPPS utilizing the new MD results is reported. We also screened libraries of inhibitors against cis-DPPS, finding ~1 μm inhibitors, and used the receiver operating characteristic-area under the curve (ROC-AUC) method to test the predictive power of X-ray and MD-derived cis-DPPS receptors. We found that one compound with potent M. tuberculosis cell growth inhibition activity was an IC(50) ~0.5- to 20-μm inhibitor (depending on substrate) of cis-DPPS, a ~660-nm inhibitor of Rv3378c as well as a 4.8-μm inhibitor of cis-FPPS, opening up the possibility of multitarget inhibition involving both cell wall biosynthesis and virulence factor formation.