11-cis-retinal and 4-azidophenylalanine

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

Topics: Azides; Cell Line; Humans; Infrared Rays; Models, Molecular; Movement; Phenylalanine; Protein Conformation; Rhodopsin; Spectroscopy, Fourier Transform Infrared; Static Electricity; Vibration

Topics: Azides; Codon, Terminator; HEK293 Cells; Humans; Phenylalanine; Protein Structure, Tertiary; Receptors, G-Protein-Coupled; Rhodopsin; RNA, Transfer; Spectroscopy, Fourier Transform Infrared

We demonstrate the site-directed incorporation of an IR-active amino acid, p-azido-L-phenylalanine (azidoF, 1), into the G protein-coupled receptor rhodopsin using amber codon suppression technology. The antisymmetric stretch vibration of the azido group absorbs at approximately 2,100 cm(-1) in a clear spectral window and is sensitive to its electrostatic environment. We used FTIR difference spectroscopy to monitor the azido probe and show that the electrostatic environments of specific interhelical networks change during receptor activation.

Topics: Azides; Electron Spin Resonance Spectroscopy; Models, Structural; Molecular Probes; Phenylalanine; Receptors, G-Protein-Coupled; Rhodopsin; Spectroscopy, Fourier Transform Infrared; Static Electricity