Conformational Changes of Cytosolic Loops of Bovine Rhodopsin During the Transition to Metarhodopsin‐ii: An Investigation by Fourier Transform Infrared Difference Spectroscopy*

Abstract

In order to assign the structural changes of the protein, observed in the Fourier transform infrared (FT-IR) difference spectra of the rhodopsin-metarhodopsin-II transition, to specific regions of the protein, rhodopsin was treated by proteases. Nonilluminated and bleached rhodopsin was treated with protease K and papain. Rhodopsin digested in the bleached state was subsequently regenerated with 11-cis-retinal. From these modified samples the rhodopsin-metarhodopsin-II FT-IR difference spectra were measured. Comparing the difference spectra with that of unmodified rhodopsin, clear deviations in the amide-I and amide-II spectral range are observed. This indicates that in the unmodified pigment conformational changes of those parts of the cytosolic surface take place which are susceptible to the proteases. From the larger spectral changes obtained with samples digested in the bleached state it is concluded that the extent of modification is larger. The difference spectra of rhodopsin modified with 10 mM dithiothreitol support the existence of the 4th loop which also undergoes conformational changes. The spectral changes are interpreted in terms of a transition of an ordered structure of the loops in rhodopsin to a more random structure in metarhodopsin-II. The results demonstrate that by combining FT-IR spectroscopy with protein modification by specific proteases, conformational changes of the protein can be localized to specific regions.

Molecular dynamics of a biophysical model for β2-adrenergic and G protein-coupled receptor activation

“…Although this picture may be more complex than previously characterized, the acid-base transition of C106 clearly creates energy perturbations that are largely compatible with many previous experimental findings [9][10][11][12][13][14][15]. In the unliganded wildtype B2AR, the TM1 Â 7, TM3 Â 4, TM3 Â 5, TM3 Â 6 and TM6 Â 7 interactions are all strongly positive for the unbound WT (B À A) states of the B2AR (gray bars in Fig.…”

“…Other investigators have also suggested that the cis-trans isomerization of retinal in rhodopsin causes repositioning followed by a movement of TM3 away from TM6 [9,11,15]. Clearly there are many studies that implicate TM3 and TM6 with the molecular events leading to receptor activation.…”

“…This suggests that one of the actions produced by EPI binding is to increase the magnitude of the TM interactions between TM1 Â 7, TM3 Â 4, TM3 Â 6 and TM6 Â 7 in the base state of the receptor. Many of these interactions have been previously linked either directly or indirectly to receptor activation [9][10][11][12][13][14][15].…”

“…Several experimental techniques show that the relative movements of transmembrane domains III and VI (TM3 and TM6) correspond with receptor activation in both the rhodopsin and B2AR receptors [9][10][11][12][13][14][15]. Gether et al, using fluorescent labels, localized agonist-induced conformational changes of the B2AR to TM3 and TM6 [12].…”

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

“…Mutations affecting cytoplasmic loops I, II, or III, or transmembrane segments (TMS) I, II, VI, or VII, constitutively activate GPCRs by destabilizing the inactive state or stabilizing the active state (Kjelsberg et al, 1992;Robinson et al, 1992;Parma et al, 1993;Robbins et al;Shenker et al, 1993;Konopka et al, 1996;Scheer et al, 1996). Indeed, conformational changes accompanying GPCR activa-tion occur in cytoplasmic loops, near the cytoplasmic terminus of TMS III or VII and within TMS VI (Ganter et al, 1992;Farahbakhsh et al, 1993;Bukusoglu and Jenness, 1996;Lin and Sakmar, 1996). Furthermore, the distance between TMS III and VI increases when rhodopsin is activated Yang et al, 1996).…”