| dc.identifier.uri | http://hdl.handle.net/1951/59665 | |
| dc.identifier.uri | http://hdl.handle.net/11401/71237 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.language.iso | en_US | |
| dc.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dc.type | Dissertation | |
| dcterms.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
Arg135<super>3.50</super> and Glu247<super>6.40</super> 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
Tyr223<super>5.58</super>, Tyr306<super>7.53</super>, Met257<super>6.40</super>,
and Arg135<super>3.50</super> 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
Tyr223<super>5.58</super> and Arg135<super>3.50</super>. Fluorescence spectroscopy is used to
measure the rate of active state decay. We find that the Tyr223<super>5.58</super> and
Arg135<super>3.50</super> 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</sub>-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 Β<sub>2</sub>-adrenergic receptor reveal that H5 rotation is a
common
element of GPCR activation. | |
| dcterms.available | 2013-05-22T17:34:36Z | |
| dcterms.available | 2015-04-24T14:46:37Z | |
| dcterms.contributor | Smith, Steven O, Scarlata,
Suzanne | en_US |
| dcterms.creator | Goncalves, Joseph Anthony | |
| dcterms.dateAccepted | 2013-05-22T17:34:36Z | |
| dcterms.dateAccepted | 2015-04-24T14:46:37Z | |
| dcterms.dateSubmitted | 2013-05-22T17:34:36Z | |
| dcterms.dateSubmitted | 2015-04-24T14:46:37Z | |
| dcterms.description | Department of Molecular and
Cellular Biology | en_US |
| dcterms.extent | 149
pg. | en_US |
| dcterms.format | Application/PDF | en_US |
| dcterms.format | Monograph | |
| dcterms.identifier | Goncalves_grad.sunysb_0771E_11045 | en_US |
| dcterms.identifier | http://hdl.handle.net/1951/59665 | |
| dcterms.identifier | http://hdl.handle.net/11401/71237 | |
| dcterms.issued | 2012-08-01 | |
| dcterms.language | en_US | |
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Previous issue date: 1 | en |
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Previous issue date: 1 | en |
| dcterms.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dcterms.subject | Biochemistry | |
| dcterms.title | Correlating Structure and Function in Class A
GPCRs | |
| dcterms.type | Dissertation | |
