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.