This is a proposal for a core facility to support a Program Project application. The main roles of this core (Core B) are to express and purify the protein targets for which anesthetic binding will be studied, and to provide structural support to the program.
Aims 1 and 2 of this core proposal focus on the production and purification of eukaryotic potassium channels;these proteins include both anesthetic-sensitive and - insensitive channels (with the latter acting as negative controls, for example for photolabeling experiments). Target proteins include the voltage-gated potassium channels Shaw2 and Kvl.2.
Aim 3 focuses on the production and purification of prokaryotic homologs of eukaryotic anesthetic targets, including the sodium channel NaChBac. All of these proteins will be expressed in heterologous systems and supplied to our colleagues, either in purified, detergent-solubilized form or as crude membrane fractions. Structural support will be supplied to the program in the form of crystallographic expertise;this expertise will be devoted to exploring crystallization conditions for the membrane proteins being studied as anesthetic targets, so as to enable future high-resolution analysis of anesthetic binding. We will also crystallize and determine structures for anesthetics bound to model proteins such as apoferritin.
|Kinde, Monica N; Bondarenko, Vasyl; Granata, Daniele et al. (2016) Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac. Proc Natl Acad Sci U S A 113:13762-13767|
|Meng, Tao; Bu, Weiming; Ren, Xianfeng et al. (2016) Molecular mechanism of anesthetic-induced depression of myocardial contraction. FASEB J 30:2915-25|
|Elokely, Khaled; Velisetty, Phanindra; Delemotte, Lucie et al. (2016) Understanding TRPV1 activation by ligands: Insights from the binding modes of capsaicin and resiniferatoxin. Proc Natl Acad Sci U S A 113:E137-45|
|Granata, Daniele; Carnevale, Vincenzo (2016) Accurate Estimation of the Intrinsic Dimension Using Graph Distances: Unraveling the Geometric Complexity of Datasets. Sci Rep 6:31377|
|Kinde, Monica N; Bu, Weiming; Chen, Qiang et al. (2016) Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels. Anesthesiology 124:664-73|
|Woll, Kellie A; Murlidaran, Sruthi; Pinch, Benika J et al. (2016) A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of Î³-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes. J Biol Chem 291:20473-86|
|Woll, Kellie A; Dailey, William P; Brannigan, Grace et al. (2016) Shedding Light on Anesthetic Mechanisms: Application of Photoaffinity Ligands. Anesth Analg 123:1253-1262|
|Carswell, Casey L; HÃ©nault, Camille M; Murlidaran, Sruthi et al. (2015) Role of the Fourth Transmembrane Î± Helix in the Allosteric Modulation of Pentameric Ligand-Gated Ion Channels. Structure 23:1655-64|
|Woll, Kellie A; Weiser, Brian P; Liang, Qiansheng et al. (2015) Role for the propofol hydroxyl in anesthetic protein target molecular recognition. ACS Chem Neurosci 6:927-35|
|Cournia, Zoe; Allen, Toby W; Andricioaei, Ioan et al. (2015) Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory. J Membr Biol 248:611-40|
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