SPECTRAL METHODS FOR STUDY OF THE G-PROTEIN-COUPLED RECEPTOR RHODOPSIN. I. VIBRATIONAL AND ELECTRONIC SPECTROSCOPY

Abstract

Here we review the application of modern spectral methods for the study of G-protein-coupled receptors (GPCRs) using rhodopsin as a prototype. Because X-ray analysis gives us immobile snapshots of protein conformations, it is imperative to apply spectroscopic methods for elucidating their function: vibrational (Raman, FTIR), electronic (UV-visible absorption, fluorescence) spectroscopies, and magnetic resonance (electron paramagnetic resonance, EPR), and nuclear magnetic resonance, NMR). In the first of the two companion articles, we discuss the application of optical spectroscopy for studying rhodopsin in a membrane environment. Information is obtained regarding the time-ordered sequence of events in rhodopsin activation. Isomerization of the chromophore and deprotonation of the retinal Schiff base leads to a structural change of the protein involving the motion of helices H5 and H6 in a pH-dependent process. Information is obtained that is unavailable from X-ray crystallography, which can be combined with spectroscopic studies to achieve a more complete understanding of GPCR function.

Keywords: FTIR spectroscopy; G-protein-coupled receptors; Raman spectroscopy; circular dichroism; electronic spectroscopy; fluorescence; linear dichroism; optical spectroscopy; rhodopsin.

Fig. 1

UV–visible spectroscopic characterization of the retinal Schiff base for the Meta I/Meta II thermal equilibrium in native disk membranes. (A, B) At 20° C and pH 5.0, the equilibrium is completely shifted to the active state of the Мета II_bH⁺ with a deprotonated Schiff base, while at 10 °C and pH 9.5 the inactive Meta I state with a protonated Schiff base is favored. (C, D) The difference spectra (photoproduct minus the dark state) demonstrate the pH-dependence of the Meta I/Meta II equilibrium at various temperatures (10 °C, 30 °C). One can see that at 30 °C the balance is not fully shifted to Meta I even at very alkaline pH. Figure is adapted from Ref. [19].

Fig. 2

Example of FTIR difference spectra of rhodopsin in the Meta I or Meta II_bH⁺ minus dark state in native disk membranes at 10 °C, pH 9.5 or 20 °C, pH 5.0, respectively. The spectral range is marked that is sensitive to conformational changes in the protein. Figure is from Ref. [19].

Fig. 3

Extended reaction scheme for rhodopsin activation in phospholipid membrane predicts a complex equilibrium of photoproducts Meta I, Meta II, and Meta II_b at alkaline pH. By contrast, at low pH the equilibrium is completely shifted to the active Мета II_bH⁺ state. Figure is adapted from Ref. [19].

Fig. 4

Examples of transient absorption and transient tryptophan fluorescence of the rod outer segment membranes after the photoactivation, showing time order sequence of changes. Note that conformational changes of rhodopsin are delayed with respect to deprotonation of Schiff base, and that subsequent proton uptake is further delayed. Conditions: pH 6, 23 °C (A) Transient absorption at 360 nm and tryptophan fluorescence of rhodopsin. Mirror image of the time-resolved absorption curve is shown by a dotted line to emphasize the similarity of the absorption and fluorescence kinetics. (B) Dependences of the absorption at 360 nm and fluorescence of Alexa594 at cysteine 316 on H8 helix. (C) Time dependences of absorption at 360 nm and 605 nm photoactivated membranes in 100 μM unbuffered solution of bromocresol purple. Figure is adapted from Ref. [57].