Type
Text
Type
Dissertation
Advisor
Smith, Steven O, Scarlata, Suzanne
Date
2012-08-01
Keywords
Biochemistry
Department
Department of Molecular and Cellular Biology
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/71237
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Class A G protein-coupled receptors (GPCRs) serve as the gatekeepers for cell signaling in eukaryotes. With over 4% of the human protein-encoding genome dedicated to their expression, GPCRs are accountable for a variety of physiological responses including, vision, vasodilation, and cell migration. These receptors all contain seven transmembrane helices and a number of conserved residues suggesting a universal activation mechanism. In order to understand GPCR activation, it is essential to delineate the structural differences between ligand-bound receptor conformations. Despite the breadth of biophysical studies conducted to date, how ligand binding is coupled to receptor activation remains to be elucidated. In this thesis, solid-state NMR studies are presented that target conformational changes in the low light visual pigment rhodopsin, a prototypical GPCR. Rhodopsin is activated by a light-induced 11-cis to trans isomerization of a covalently bound retinal chromophore. The experimental data presented define global structural changes that couple receptor activation with the binding of downstream signaling targets. Activation-induced changes are described in the region of transmembrane helices H5 and H6. First, NMR distances measurements are used to temporally separate the motion of H6. Using 13C...13C dipolar couplings we observe a rotation of transmembrane helix H6 upon formation of Meta I. Meta I is the inactive predecessor of the signaling competent state, Meta II. Rotation of H6 in Meta I reflects the disruption of a salt bridge between Arg1353.50 and Glu2476.40 prior to displacement of the transmembrane helix in Meta II, which is required for coupling to heterotrimeric G protein. In addition, we show that H5 undergoes a rotation in the transition to Meta II. Specifically, we observe NMR contacts between Tyr2235.58, Tyr3067.53, Met2576.40, and Arg1353.50 that reveal a close association between these residues in the active Meta II state. Rotation of H5 allows a direct interaction to form between signature-conserved residues Tyr2235.58 and Arg1353.50. Fluorescence spectroscopy is used to measure the rate of active state decay. We find that the Tyr2235.58 and Arg1353.50 interaction is crucial in stabilizing the active conformation of H5. The structural studies on rhodopsin are extended to the ligand-activated Β#60;sub>2-adrenergic receptor. We use NMR to probe the rotational orientation of transmembrane helix H5 in the presence of various ligands. The data show a graded rotation of H5 that correlates with ligand efficacy. Together, the structural studies on rhodopsin and the Β2-adrenergic receptor reveal that H5 rotation is a common element of GPCR activation. | 149 pages
Recommended Citation
Goncalves, Joseph Anthony, "Correlating Structure and Function in Class A
GPCRs" (2012). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 443.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/443