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Description

Proteins are three-dimensional structures of linear polymers of amino acids; the direct outcomes of genetic blueprints of all living things and essential to all biological processes. One such class of proteins is voltage-gated ion channels (VGICs), integral membrane proteins that form gateways within cell membranes permitting the selective transport of VGIC-specific ions across the membrane in response to changes in membrane potential. Voltage gated potassium channels (K[subscript V], VGKC) represent a large family of membrane-embedded channels highly selective for potassium ions (K+). Consisting of four identical subunits each with six transmembrane segments (S1-S6), and an ion-specific selectivity filter (SF), K[subscript V] channels reside in electrically excitable neuronal, muscle, and endocrine cells, where they are crucial for membrane repolarization and regulation of the outward K+ flow. In this work, we apply Rosetta computational modeling and design to study structure, gating, and function of two VGKCs: K[subscript V]11.1 and K[subscript V]2.1. We first delve into the structural modeling of the inactivated-state of K[subscript V]11.1, encoded by the human Ether-à-go-go-Related Gene (hERG). The hERG channel is a VGKC found primarily in cardiac myocytes where it regulates the repolarization phase of the ventricular action potential (AP). This process is associated with hERG's rapid, voltage-dependent, C-type inactivation, which blocks ion conduction and is suggested to involve constriction of the selectivity filter. hERG is implicated in a number of congenital and drug-induced arrhythmias, caused by long QT syndrome, which may result from defective C-type inactivation gating kinetics as well as disruption of ion conduction from drug binding in the channel pore. Mutations S620T, S641A/T, and G648A, in and around the selectivity filter region of hERG have been shown to alter the voltage-dependence of channel inactivation. To explore conformational changes associated with hERG inactivation, we use the Rosetta computational modeling software Relax application to simulate the structural effects of those mutations and how they can modulate channel gating, and Rosetta GA-LigandDock to explore the state-dependent binding mechanism of dofetilide, terfenadine, and E4031; highly selective and potent hERG blockers with known pro-arrhythmia risks. We show that the S641A fast-inactivating mutation enables conformational change resulting in a fenestration region below the selectivity filter similar to "hydrophobic pockets" in the WT cryo-EM structure, and a lateral shift of residue F627 in the selectivity filter into the central channel axis along the ion conduction pathway. Non-inactivating mutations S620T and S641T showed a potential blocking mechanism of F627 rearrangement, preventing it from shifting into the conduction pathway during the proposed inactivation process. Additionally, drug docking results correlate well with existing experimental evidence of protein-ligand contacts between high-affinity hERG blockers and key residues Y652 and F656 inside the pore cavity, in addition to illuminating potentially new ligand binding interactions in the inactivated state fenestration region. The goal of the second project was to rationally design a genetically encoded fluorescent reporter of neuronal activity, with the intent to probe activation of the neuronal voltage-gated potassium channel, K[subscript V]2.1, which is widely expressed in the brain and regulates neuronal excitability and action potential duration. For our fluorescent reporter of voltage activation, we use the E. coli ethidium bromide multi-drug binding protein (EbrR), a natural binder of malachite green (MG) which elicits a strong fluorescent signal upon binding. Rosetta design and ligand docking methods were then used to develop a variant of EbrR using a non-membrane permeable MG analog, malachite green [beta]-alanine (MGBA). We describe the development of a protocol for recombinant expression and protein purification of EbrR, and demonstrate that MGBA binds similarly to MG, with strong [pi]-[pi] interactions to key hydrophobic residues, eliciting a fluorescence signal. Additionally, Rosetta design results indicate an increase in binding stability of MGBA for mutant EbrR variants, with potential for increasing fluorescence output. Taken together, we demonstrate the utility of Rosetta in advancing our knowledge of the conformational changes associated with hERG channel inactivation and associated mutations and provide a foundation for the development of a fluorescence reporter of voltage activation of K[subscript V]2.1 channel. The use of Rosetta structural modeling software marks these studies as part of a multidisciplinary approach to provide atomic-level structural understanding of conformational changes related to channel gating and develop molecular tools to visualize channel activity in excitable cells.

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