• Advancing Drug and Biomaterial Design with Constant pH Molecular Dynamics Simulations

      Tsai, Cheng-chieh; Shen, Jana (2019)
      Molecular dynamics (MD) simulation is a valuable tool for investigating motions of macromolecular systems; however, solution pH, a critical factor for biological and chemical processes, is neglected in conventional MD simulations. To address this weakness, our group developed various continuous constant pH molecular dynamic (CpHMD) tools. In this dissertation, applications and new protocols that utilize CpHMD for drug and biomaterial design are presented. β-secretase 1 (BACE1, implicated in Alzheimers disease) and Src kinase (implicated in cancer) were utilized as model systems for drug design applications. In chapter 2, we applied CpHMD to understand the binding of BACE1 with two small-molecular inhibitors. We discovered that, despite the structural similarity of the two inhibitors, the titration behavior of the protein active site differs and this difference dramatically impacts the protein-ligand interactions and consequently the inhibitor affinity. Further, we tested a new protocol that combines CpHMD titration with free energy simulations to construct the pH-dependent binding free energy profiles. The resulting data showed excellent agreement with experiment and identified one potential allosteric site in BACE1. Next, in chapter 3, we simulated Src kinase to test the capability of CpHMD tostudy kinases. Starting from the crystal structure of the inactive Src, CpHMD can capture the conformational activation along the major inactive states without introducing any biasing potential or mutation. Starting from chapter 4, we studied the chitosan-based hydrogel systems to explore the detailed molecular mechanisms that give rise to macroscopic materials properties. We examined the flexibility of chitosan chains under different environmental conditions such as pH and salt concentration. Simulation data revealed that in addition to electrostatic screening, salt ions enhance the chain flexibility by interrupting the intra-molecular hydrogen bonds and thereby shifting the conformational populations between extended and bent states. In chapter 5, the atomic-level mechanism of pH-responsive chitosan-based hydrogels with switchable mechanical properties was investigated. Our data suggested that the electrostatic crosslinks are formed through the pH-dependent salt-bridge interactions between the chitosan glucosamines and surfactant headgroups. The pKa difference between the chitosan crystallite and the surfactant-bound chitosan is a key for the persistent but erasable gradient in the structural and mechanical properties between the two crosslinked regions. In summary, my work provids insights that will contribute to drug and biomaterial design and highlights the usefulness of CpHMD in these fields.
    • Development of Additive and Polarizable Force Field Parameters for Polypeptides

      Zhu, Xiao; MacKerell, Alexander D., Jr. (2012)
      Simulations of biological macromolecules are continually improving due to the extension and continued optimization of empirical force fields based on molecular mechanics. The quality of this "balls-on-springs" model is likely to increase, though limited by the nature of molecular mechanics, as more experimental data become available allowing for further optimization of the underlying parameters. Classically, force fields employed a fixed-charge, or additive, scheme where partial atomic charges on atoms cannot change during a simulation such that changes in molecular dipole (i.e. explicit polarization) as a function of environment are lost. Here, we describe the development, optimization, and application of a polarizable CHARMM force field that explicitly accounts for molecular polarizability via the inclusion of atomic polarizability based on the classical Drude oscillator. We begin by presenting an overview of the history of the CHARMM empirical force field, its various components, applications, as well as the formalisms for the inclusion of polarizability in force fields. This is followed by development efforts as part of this thesis towards the completion of the polarizable force field for simulations of protein. In Chapter 3, a systematic study of the side-chain conformations was performed using quantum mechanical methods. Through comparisons with a large-scale survey of the protein crystal structures, a relationship was drawn between the intrinsic energetics and occurrences in protein structures. The intrinsic energetics were then used as target data for the optimization of side-chain torsion parameters, as discussed in Chapter 4; the data and optimization approach is likely to be of value to other empirical force field development communities. Because one of the advantages of the polarizable force field is more accurate description of the electrostatics, the performance of the force field in the calculation of pKas was undertaken. In Chapter 5 we present data for the development of parameters for alternative protonation states of the Cys, Lys, and Tyr side-chains, opening doors to simulations of these species within protein systems. Finally, in Chapter 6 we applied the polarizable force field in the calculation of pKas for select residues in RNase A, demonstrating the advantages and potential challenges with respect to future optimization of the Drude polarizable force field.
    • Exploring Protein Dynamics and Folding with Constant pH Molecular Dynamics Simulations

      Yue, Zhi; Shen, Jana; 0000-0002-4231-7474 (2017)
      Solution acidity or pH is a key environmental regulator with profound impact on biological processes such as protein dynamics and functions. Over the past decade, our research group has developed a set of tools to explicitly account for solution pH in molecular dynamics simulations. In this dissertation we demonstrate the new application areas of the constant-pH molecular dynamics tools. First we use constant-pH MD alongside fixed-protonation-state simulations to explore the dynamics of a cytoplasmic heme-binding protein (PhuS) in Pseudomonas aeruginosa, a notorious opportunistic pathogen. Our results provide atomic-level information on how heme binding affects PhuS dynamics which suggests an induced-fit mechanism, in support of the recent hydrogen-deuterium exchange data. Secondly, we apply constant-pH MD to investigate the acid-induced unfolding of BBL, a small component of the ubiquitous pyruvate dehydrogenase multienzyme complex involved in carbohydrate metabolism inside mitochondria. Our data support that BBL is a barrier-limited two-state folder, an issue that has drawn intense debate among experimental groups. More importantly, our simulations reveal that acid-induced unfolding of BBL is triggered by sequential protonation of His166 and Asp162 and thereby offering atomic details unattainable via experimental means. This work is the first time the constant-pH MD in explicit solvent has been applied to protein folding studies. Next, we employ the membrane-enabled constant-pH MD to understand how proton release drives the conformational transition of the transmembrane multi-drug efflux pump AcrB, which is crucial for the intrinsic resistance of E. coli to clinically important antibiotics. Our data address the controversy regarding the proton/drug stoichiometry and reveal the details of how deprotonation of a single residue leads to a global conformational transition in AcrB. This work paves the way for understanding the complete cycle of drug transport in AcrB and validates the membrane-enabled constant-pH MD technique for mechanistic studies of proton-coupled transporters. Lastly, we benchmark the accuracy of the all-atom constant-pH MD with charge-leveling co-ion using titration simulations of five proteins (HP36, BBL, NTL9, HEWL and SNase). The average and maximum absolute errors between the calculated and experimental pKa values based on 10-ns pH-based replica-exchange simulations are 0.7 and 0.9 units, respectively. Detailed analyses indicate that limited sampling is a major source of error. This work demonstrates the all-atom constant-pH MD method a practical tool for accurate prediction of pKa's and atomically detailed studies of proton-dependent conformational dynamics. In summary, my studies offered new mechanistic insights into the various roles of protons in protein dynamics and folding that were previously not well understood. My work further established constant-pH MD as a powerful tool for revealing atomic details of proton-coupled dynamic processes.