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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM055876-15
Application #
8740501
Study Section
Special Emphasis Panel (ZGM1-PPBC-5)
Project Start
Project End
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
15
Fiscal Year
2014
Total Cost
$211,206
Indirect Cost
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Yang, Elaine; Granata, Daniele; Eckenhoff, Roderic G et al. (2018) Propofol inhibits prokaryotic voltage-gated Na+ channels by promoting activation-coupled inactivation. J Gen Physiol 150:1299-1316
Woll, Kellie A; Guzik-Lendrum, Stephanie; Bensel, Brandon M et al. (2018) An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules. J Biol Chem 293:11283-11295
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Kasimova, Marina A; Yazici, Aysenur Torun; Yudin, Yevgen et al. (2018) A hypothetical molecular mechanism for TRPV1 activation that invokes rotation of an S6 asparagine. J Gen Physiol 150:1554-1566
Wang, Yali; Yang, Elaine; Wells, Marta M et al. (2018) Propofol inhibits the voltage-gated sodium channel NaChBac at multiple sites. J Gen Physiol 150:1317-1331
Woll, Kellie A; Skinner, Kenneth A; Gianti, Eleonora et al. (2017) Sites Contributing to TRPA1 Activation by the Anesthetic Propofol Identified by Photoaffinity Labeling. Biophys J 113:2168-2172
Bensel, Brandon M; Guzik-Lendrum, Stephanie; Masucci, Erin M et al. (2017) Common general anesthetic propofol impairs kinesin processivity. Proc Natl Acad Sci U S A 114:E4281-E4287
Okuno, Toshiaki; Koutsogiannaki, Sophia; Ohba, Mai et al. (2017) Intravenous anesthetic propofol binds to 5-lipoxygenase and attenuates leukotriene B4 production. FASEB J 31:1584-1594
Granata, Daniele; Ponzoni, Luca; Micheletti, Cristian et al. (2017) Patterns of coevolving amino acids unveil structural and dynamical domains. Proc Natl Acad Sci U S A 114:E10612-E10621

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