Student Theses and Dissertations


Tian He

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Sakmar Laboratory


G protein-coupled receptors (GPCRs) constitute a large family of transmembrane receptors that transduce extracellular signals into intracellular biochemical responses. Understanding with chemical precision how GPCRs function in cellular membranes is an active area of biological research, but despite recent reports of X-ray crystal structures of several GPCRs, some important questions remain unresolved. For example, the kinetics and thermodynamics of ligand-receptor interactions that lead to receptor activation and how allosteric modulators affect receptor signaling need to be addressed. In addition to basic understanding of transmembrane signaling, studies of GPCRs can provide insights that might advance drug discovery since a large proportion of existing therapeutic agents target GPCRs. The first aim of my thesis project was to develop a strategy for the bioorthogonal labeling of GPCRs with useful chemical probes or chemically reactive handles at specific defined sites. Several strategies were explored to label unnatural amino acid residues genetically encoded into GPCRs expressed in mammalian culture. Using the visual pigment rhodopsin (Rho) as a model GPCR, the strain-promoted [3+2] azide-alkyne cycloaddition reaction (SpAAC) between dibenzocyclooctyne (DIBO) and p-azido-L-phenylalanine (azF) was shown to be a suitable strategy for attaching labels to GPCRs. While characterizing the specificity, kinetics, topology-dependent reactivity of the labeling chemistry, the reaction rate of SpAAC with azF situated in the transmembrane region of the receptor was enhanced by up to 1000-fold, which was attributed to DIBO partitioning into the hydrophobic core of micelles. Then a fluorescence resonance energy transfer (FRET) assay was developed for the labeled Rho to demonstrate its functionality with respect to ligand binding. Rho consists of a chromophore ligand, 11-cis-retinal, bound to the opsin via a Schiff base bond. The photoisomerization of 11-cis-retinal to all-trans-retinal activates the receptor to trigger the downstream signaling cascade in photoreceptor cells. The Schiff base bond in the active conformation is prone to hydrolysis, allowing all-trans-retinal to dissociate from the ligand-binding pocket. The ligand-free opsin then recombines with 11-cis-retinal to complete the visual cycle. The FRET assay used to measure the reaction rates of bioorthogonal labeling chemistries also enabled measurement of retinal entry kinetics and was utilized first to address the energetics of the recombination reaction between opsin and 11-cis-retinal. The activation energy for retinal binding was obtained from the temperature-dependent retinal entry kinetics. The reaction enthalpy was measured by isothermal titration calorimetry (ITC). The activation energy for the reverse reaction was measured by chromophore exchange of the bound 11-cis-retinal with exogenous 9-cisretinal. Based on these results, the complete energy diagram was derived for the binding between 11-cis-retinal and opsin. Additional studies were carried out to determine how retinal entry and release kinetics were affected by site-directed mutagenesis of set of highly conserved amino acid residues postulated to be in the pathway for retinal entry and/or release. A set of mutations located at the fifth and sixth transmembrane (TM) helices was found to exert a much greater influence on the retinal entry kinetics than on the retinal release kinetics. Three criteria were used to evaluate the influence of these mutations: 1) the correlation between side-chain size and entry kinetics; 2) the change in the activation energy for retinal entry; 3) the effect of increasing polarity at these sites. Based on these findings, a model was proposed to describe the retinal entry pathway leading from the membrane-embedded receptor surface to the ligand-binding site in the transmembrane core. In summary, the methods described in this thesis add to the chemical biology tool kit for probing the structure-function relationship in GPCRs. As a specific application of the methodology a detailed analysis is presented that describes the kinetics and thermodynamics of ligand binding and release in the prototypical GPCR rhodopsin. Additional applications of the current methodology include, for example, single-molecule studies of the GPCR signaling complex.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirement for the degree of Doctor of Philosophy

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