FTIR analysis of GPCR activation using azido probes.
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.
Ye
Shixin
S
Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York, USA.
Huber
Thomas
T
Vogel
Reiner
R
Sakmar
Thomas P
TP
D001386:Azides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008961:Models, Structural; D015335:Molecular Probes; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D055672:Static Electricity
D001386:Azides; D015335:Molecular Probes; D043562:Receptors, G-Protein-Coupled; C027162:4-azidophenylalanine; D010649:Phenylalanine; D012243:Rhodopsin