Fluorescence spectroscopy of rhodopsins: Insights and approaches

• Published in: Biochimica et biophysica acta 2014-05, Vol.1837 (5), p.694-709
• Main Authors: Alexiev, Ulrike, Farrens, David L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cattle; Fluorescence Polarization; Fluorescence spectroscopy; Fluorescent Dyes - chemistry; Humans; Models, Molecular; Molecular Sequence Data; Photons; Protein Structure, Secondary; Protein Structure, Tertiary; Retinal protein; Retinaldehyde - chemistry; Retinaldehyde - metabolism; Rhodopsin - chemistry; Rhodopsin - metabolism; Site-directed fluorescence labeling; Spectrometry, Fluorescence - methods; Spectrometry, Fluorescence - statistics & numerical data; Visual rhodopsin
• ISSN: 0005-2728; 0006-3002; 1879-2650
• DOI: 10.1016/j.bbabio.2013.10.008

Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks. •Fluorescence theory, methods and analysis used to study rhodopsins are reviewed.•Retinal makes rhodopsin both a challenge and an opportunity for fluorescence studies.•Challenges include potential sample bleaching, and FRET of fluorophore to retinal.•Visual rhodopsin studies have given novel insights into GPCR structure and dynamics.