Molecular Dynamics Simulations Of Biological Macromolecules

National Heart, Lung, And Blood Institute

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Abstract

pH dependance of a Na channel Sodium channels play an important role in electrical signaling in cells; as such they are the targets of many drugs, as well as naturally occurring toxins from plants and animals. Inhibition and/or improper functioning of sodium channels due to mutation can lead to disease. In bacterial voltage gated sodium channels, the passage of sodium ions through the pore is controlled by a selectivity filter (SF) comprised of four glutamate residues for the bacterial channel NavMs. Previously, we have shown with MD simulations at constant pH and with free energy perturbation that the pKa values of the four SF glutamate residues depend on the number of ions bound in the channel, and that the fully deprotonated, singly protonated and doubly protonated states of the SF are all possible at physiological pH. Based on the MD simulations of the fully open channel, we have further shown that the conductance of the channel decreases with each proton bound to the SF. Thus the conductance of the channel is pH dependent, and decreases with lowering of pH, in agreement with experiments on similar channels. We also show that the conductance depends on the lipid composition of the membrane. Identify the conformational states of glycine receptor alpha3 through umbrella sampling Ligand-gated ion channels allows cells to respond rapidly to changes in their external environment. The structure change from one state to other states is the key to understand how ion channels function. In many cases due to the limitation in experiment, only some state structures, a closed, open state, or desensitized state, are available. A reliable method is needed to derive the structures of the missing state structures. Even in cases structures of all states are available, it is desired to understand how conformation changes during state transition. We present a method that utilizes the umbrella sampling to drive conformation changes from one state to the other state. A reaction coordinate describing relative orientation of lining substructures of ion channels is proposed and the free energy profile along the reaction coordinates is produced. For glycine receptor alpha-3 pentamer, we find that there are two free energy wells separated by a barrier in the free energy profile. The desensitized state corresponding to one well at a larger reaction coordinate and the closed state corresponding to the other well at a smaller reaction coordinate. The open state locates at the barrier region and the high free energy of the open state make it unstable, which is a main reason that the open state is difficult to be captured in experiment. This free energy profile also explains the observation that the open state structure quickly decays to the other states in some computational studies. Examining the conformations of these state shows that glycine binding produces an expansion movement at the ECD and TMD interface, which opens the ion gate at the middle of the ion channel. Continued opening will result in the structure decaying into the free energy well of the desensitized state. At the desensitized state, the proline residues at bottom of the ion channel close to lock up the channel. The result agrees with the close, open, and desensitized state structures of the glycine receptor alpha1. The free energy profile along the reaction coordinate provides a thermodynamic understanding of the ion channel functional states. Conformational fluctuations in 2-microglubulin using Markov state modeling and molecular dynamics Conformational dynamics in proteins can give rise to aggregation prone states during the folding and these kinetically stable states could form oligomers and aggregates. We investigated the intermediate states and near folded states of 2-microglobulin using molecular dynamics and Markov state modeling. Analysis of hundreds of microseconds simulation showed the importance of the edge strands A and D in the misfolded states that give rise to a high exposure of hydrophobic residues in the core of the protein that could initiate oligomerization and aggregate formation. Our Markov model showed that fluctuations in the structure of 2m that were in the order of 10s of s. Our study shed light on the first step of aggregation of 2m monomers and gave a better picture of the landscape of protein misfolding. Structure analysis and simulation study of microtubule dynamics Microtubules are a structurally and functionally important components of the eukaryotic cytoskeleton that play a crucial role in cell division and intracellular trafficking. Understanding of the mechanism of microtubule dynamics is crucial to control cell proliferation. Disrupting microtubule dynamics is a major strategy in cancer therapy. Through structure analysis of tubulin conformations in microtubules and in unassembled forms, we discovered a rotation of the tubulin intermediate domain that switches tubulins from a closed state to an open state. This rotation is responsible for the conformational change of tubulins during microtubule growth. Based on the observation that a GDP shift coincident with the rotation; I propose a hypothesis that the GTP hydrolysis produces a GDP stroke that causes the rotation. Through self-guided Langevin dynamics simulations of tubulin monomers and heterodimers, with and without the GDP stroke, this work proves that the GDP stroke does cause the rotation. At the closed state, tubulins polymerize into a curved protofilament. In the open state tubulins can dock into the open pockets to form a straight protofilament. Lateral interactions between straight protofilaments stabilize microtubules. Based on these results, this work proposes a hydrolysis driven mechanism that can well describe microtubule dynamics. This new mechanism will facilitate new strategies in regulating microtubule dynamics and in development of cancel therapy. Molecular dynamics of ligand binding to PAS domain of EAG channel We investigated ligand binding to the PAS domain of EAG potassium channel and its effect on the channel activity using MD simulations, free energy calculations and network analysis. We performed docking of ligands to the PAS domain of EAG channel and found a residue Tyr71 that was blocking the entrance to the binding pocket. Replica exchange simulations allowed us to sample conformations of PAS where the Tyr71 shifted away from binding pocket and allowed docking of small ligands to the PAS domain. Binding free energy calculation using MMPBSA showed a favorable binding of ligands to the PAS domain which was majorly driven by hydrophobic interaction. Furthermore, our network analysis on the trajectories of full length EAG channel in the bound state showed that there is an allostery between conformational fluctuations in PAS and CNBHD and the pore residues. We used information flow analysis to find if there is an allosteric pathway between the ligand binding site and the channel pore and regions and residues along the pathway that carry most of the information flow. Our analysis showed that CNBHD carries most of information flow from PAS to channel pore. Moreover, the channel residues had a lower current flow in the bound state. Since the simulated protein is in the inactive state, this implies a further stabilization of the closed state of the EAG channel.