Ftir Studies On The Activation Mechanism Of Rhodopsin; Possible Extension To General Gpcr'S

The visual pigment rhodopsin belongs to the group of GPCR's and is so far the best characterized member. Although the structure is known since 2000, the activation mechanism is still not completely understood. Activation is triggered by the light-induced conversion of the covalently bound inverse agonist, 11-cis retinal, to the all-trans geometry, constituting an agonist. We have studied the activation mechanism by Fourier transform infrared difference spectroscopy, a technique capable of monitoring molecular changes of proteins and co-factors. Therefore, this technique could also be of importance for the study of general GPCR's. Some of our recent results will be reviewed. They include the study of the retinal-free opsin state, which can be activated by low pH. This observation has been relevant for the recently published structures of opsin. Next, we show how the technique can be used to study conformational changes of the active state induced by the interacting G-protein (here a peptide derived from the Gα C-terminus known to be responsible for the interaction). In addition, the technique reveals conformational changes of the peptide. Further, the different ionic locks keeping the receptor in its inactive state are addressed. They encompass the Schiff base-counterion, the ERY motif at the end helix C, and the interaction of this motif with two further glutamic acids on helix F. This interaction is thought to keep also the β-adrenergic receptor in its inactive conformation. Finally, a method will be proposed which will allow the study of the ligand-induced activation of general GPCR´s. The essential feature is a receptor monolayer, allowing free access of the ligand. The required increase in sensitivity is realized by surface-enhanced infrared spectroscopy.