Elucidating allosteric signal transmission in the beta1-adrenergic receptor
Third-party funded project
Project title Elucidating allosteric signal transmission in the beta1-adrenergic receptor
Principal Investigator(s) Isogai, Shin
Project Members Hartmann, Jean-Baptiste
Delgado, Leonildo
Organisation / Research unit Departement Biozentrum / Structural Biology (Grzesiek),
Departement Biozentrum / Structural Biology (Isogai)
Project start 01.04.2017
Probable end 31.08.2019
Status Completed
Abstract

G protein coupled receptors (GPCRs) are an important class of trans-membrane proteins that

recognize a multitude of extracellular molecules and transmit their signal to the intracellular side.

Despite recent achievements in X-ray crystallography of GPCRs, the high-resolution structures

obtained do not capture their intrinsic dynamic properties, which are tightly associated with their

function. NMR spectroscopy promises to provide such missing dynamic information. However so far,

despite being valuable, only limited information has been obtained by NMR due to the difficult

spectroscopic properties of this protein class. At this point, the potential of solution NMR analysis of

GPCRs has not been fully realized.

Recently, I have overcome many of the obstacles that hinder the application of solution NMR to

study signal transduction in GPCRs. Using a stabilized form of the β1 -adrenergic receptor and a

selective isotope labeling method in the baculovirus-insect cell expression system, I was able to

acquire well-resolved backbone amide proton-nitrogen correlation spectra. These spectra revealed

numerous mechanisms within the receptor that are new, or had been postulated but never observed

directly. Thus I have established a system that can now be used to study many more functional

mechanisms of GPCRs at atomic resolution. In addition, we have developed an economic method to

produce uniformly isotope-labeled (including deuteration) GPCRs in the insect cell systems. This will

allow more advanced applications of NMR spectroscopy to GPCRs such as the study of their dynamics

by relaxation measurements.

In the present proposal I want to use this system to obtain detailed insights into the receptor’s signal

transmission mechanism with the aim to understand how the receptor recognizes ligands and passes

this information to the G protein in order to modulate its activity. Using state-of-the-art methods of

isothermal titration calorimetry, protein rigidity theory, coevolutionary sequence alignment, in vitro

real-time observation of GDP/GTP exchange by fluorescence as well as solution NMR, I want to

elucidate the underlying molecular mechanism of the thermodynamic behavior of ligand-receptor

interactions, determine a high-resolution allosteric network model of signal transmission, and provide

mechanical insights into how different agonists elicit varying levels of G protein activation.

If successful, the results will have implications for the general understanding of GPCR function and

the developed methods should be applicable to other GPCRs.

Keywords G protein coupled receptor, protein allostery, nuclear magnetic resonance, thermodynamics, in vitro fluorescent kinetic analysis
Financed by Swiss National Science Foundation (SNSF)
   

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