NDLI: A Constitutively Activating Mutation Alters the Dynamics and Energetics of a Key Conformational Change in a Ligand-free G Protein-coupled Receptor

Content Provider	Journal of Biological Chemistry (JBC)
Author	Tsukamoto, Hisao Farrens, David L.
Abstract	G protein-coupled receptors (GPCRs) undergo dynamic transitions between active and inactive conformations. Usually, these conversions are triggered when the receptor detects an external signal, but some so-called constitutively activating mutations, or CAMs, induce a GPCR to bind and activate G proteins in the absence of external stimulation, in ways still not fully understood. Here, we investigated how a CAM alters the structure of a GPCR and the dynamics involved as the receptor transitions between different conformations. Our approach used site-directed fluorescence labeling (SDFL) spectroscopy to compare opsin, the ligand-free form of the GPCR rhodopsin, with opsin containing the CAM M257Y, focusing specifically on key movements that occur in the sixth transmembrane helix (TM6) during GPCR activation. The site-directed fluorescence labeling data indicate opsin is constrained to an inactive conformation both in detergent micelles and lipid membranes, but when it contains the M257Y CAM, opsin is more dynamic and can interact with a G protein mimetic. Further study of these receptors using tryptophan-induced quenching (TrIQ) methods indicates that in detergent, the CAM significantly increases the population of receptors in the active state, but not in lipids. Subsequent Arrhenius analysis of the TrIQ data suggests that, both in detergent and lipids, the CAM lowers the energy barrier for TM6 movement, a key transition required for conversion between the inactive and active conformations. Together, these data suggest that the lowered energy barrier is a primary effect of the CAM on the receptor dynamics and energetics.
Related Links	http://www.jbc.org/content/288/39/28207.abstract
Ending Page	28216
Starting Page	28207
Page Count	10
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	39
Volume Number	288
DOI	10.1074/jbc.M113.472464
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2013-09-27
Access Restriction	Open
Subject Keyword	Fluorescence G Protein-coupled Receptors (GPCR) Protein Dynamics Receptor Structure-Function Rhodopsin Conformational Transitions Energy Landscapes Protein Energetics Site-directed Fluorescence Labeling (SDFL) Tryptophan-induced Quenching (TrIQ) Signal Transduction
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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