Project 3 will study anesthetic effects on protein global dynamics and test the central hypothesis that only those binding sites at which anesthetic binding can cause global dynamics changes are essential for mediating anesthetic action on proteins. Solution-state NMR in lyso-lipid micelles and high-resolution magicangle spinning (MAS) NMR in low-q lipid bicelles will be used to study a member ofthe Cys-loop ligandgated ion channel (LGIC) family (specific aim #1) and a member ofthe voltage-gated ion channel (VGIC) family (specific aim 2). We will focus on the transmembrane (TM) domain of nAChR a7 subunit in the functional homopentameric form with and without the intracellular (IC) domain, and the bacterial voltagegated sodium channel NaChBac. An anesthetic binding site will be engineered near the TM2-TM3 linker in nAChR a7 TM domain to turn this inhaled anesthetic-insensitive channel into an anesthetic sensitive channel. High-resolution TM domain structure of nAChR-a7 and the voltage-sensing domain (VSD), the pore domain (PD), and the 84-85 linker of NaChBac will be determined. Strategic ^^F probe placements, one or a few at a time, will be implemented to selectively label Phe, Lys, or site-directed Cys mutations to generate structural constraints. A model-based structure refinement approach will be taken through iterative interaction with Project 4 and Project 5 to refine the full-length receptor structures in the functional forms. The same ^?F labels will also be used to determine slow domain dynamics through ^^F T2 dispersion measurements in the solution state. To complement the photoaffinity binding analyses in Project 1, intermolecular ^^F-^?F NOESY and ^^F-^H HOESY between fluorinated anesthetics (halothane, isoflurane, sevoflurane, and fluorinated propofol) and the ^^F labels at various sites in the proteins will be used to determine anesthetic binding sites and affinities. We will classify and characterize at least two types of anesthetic binding sites: Type I where anesthetic binding can cause global dynamics changes and Type II where anesthetic binding produces only local effects. Through interaction with Project 2, we will seek correlations between anesthetic binding at Type I versus Type II sites and the effects of anesthetic binding on channel function. This project will provide new mechanistic insights into the """"""""down-stream events"""""""" of anesthetic binding in modulating changes in channel activation and function.

Public Health Relevance

This and other projects determine character of drug binding sites on relevant proteins, and thereby enable drug improvements. More importantly, this project links the binding site character with the functional role, allowing the assignment of importance to sites, so that we know which binding site character to design our drugs for.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM055876-15
Application #
8740497
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
$300,446
Indirect Cost
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Granata, Daniele; Carnevale, Vincenzo (2016) Accurate Estimation of the Intrinsic Dimension Using Graph Distances: Unraveling the Geometric Complexity of Datasets. Sci Rep 6:31377
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
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
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

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