Engineering, Large Scale Expression, and NMR Spectroscopy of the Human Beta1-Adrenergic Receptor

Abstract

G-Protein coupled receptors (GPCRs) are membrane proteins, conveying an extracellular stimulus over the plasma membrane with an intracellular response as a consequence. These receptors are found in all eukaryotic organisms, and particularly in higher eukaryotes they play crucial roles, for example, regulating heart rate, transmitting signals in neural synapses in the brain, or enabling vision. Being the targets of an estimated 30% of all pharmaceutical drugs, their importance can hardly be overstated. Only recently, 3-dimensional structures have become available, facilitating structure-based drug design. To a large extent, however, information on conformational changes when a GPCR is activated or antagonized is lacking. In addition, dynamical characterization is also absent. An ideal tool to study this, conformational dynamics of proteins, is nuclear magnetic resonance (NMR) spectroscopy. Here, an engineered and thermo-stabilized version of the human beta1- adrenergic receptor has been optimized for structural studies, improving the yield of the receptor in eukaryotic expression systems, while retaining thermo- stability, making NMR studies more feasible in terms of labor and finances. This was done through testing an array of modifications on the protein level. In addition, expression conditions using the baculovirus-insect cell system have been optimized to yield protein of higher quality, an improvement which is most likely generally applicable to membrane protein over-expression. Likewise, an optimized procedure for fast purification of receptor from large-scale expression was set up. The purified protein exhibited biophysical characteristics, resembling those of the avian model GPCR, the turkey beta1- adrenergic receptor, which is known to readily crystallize and is amenable to NMR studies. Finally, NMR experiments were carried out with the human beta1- adrenergic receptor, resulting in 2-dimensional [1H,15N]-TROSY spectra, of very similar appearance to those previously obtained for the turkey beta1- adrenergic receptor. Most interestingly, a spectrum with the clinically important beta-blocker S-propranolol, which has also been described as a biased agonist, was obtained, which showed that this ligand induces a unique state of the receptor – an effect not seen for the previously tested antagonist on the avian homolog. This thesis demonstrates that NMR studies of an extremely challenging GPCR, are indeed possible, but also clearly shows that it is a formidable task that is still in need of additional optimization before the full potential of NMR spectroscopy on this type of protein can be unleashed.