Rhodopsin in Nanodiscs Has Native Membrane-like Photointermediates

Abstract

Time dependent studies of membrane protein function are hindered by extensive light scattering that impedes application of fast optical absorbance methods. Detergent solubilization reduces light scattering but strongly perturbs rhodopsin activation kinetics. Nanodiscs may be a better alternative if they can be shown to be free from the serious kinetic perturbations associated with detergent solubilization. To resolve this, we monitored absorbance changes due to photointermediates formed on the microsecond to hundred millisecond time scale after excitation of bovine rhodopsin nanodiscs and compared them to photointermediates that form in hypotonically washed native membranes, as well as to those that form in lauryl maltoside suspensions at 15 and 30 °C over a pH range from 6.5 to 8.7. Time-resolved difference spectra were collected from 300 to 700 nm at a series of time delays after photoexcitation, globally fit to a sum of time-decaying exponential terms, and the photointermediates present were determined from the spectral coefficients of the exponential terms. At the temperatures and pHs studied, photointermediates formed after photoexcitation of rhodopsin in nanodiscs are extremely similar to those that form in native membrane, in particular displaying the normal forward shift of the Meta I480 ⇄ Meta II equilibrium with increased temperature and reduced pH which occurs in native membrane but which is not observed in lauryl maltoside detergent suspensions. These results were obtained using the amount of rhodopsin in nanodiscs which is required for optical experiments with rhodopsin mutants. This work demonstrates that late, physiologically important rhodopsin photointermediates can be characterized in nanodiscs, which provide the superior optical properties of detergent without perturbing the activation sequence.

Rod Visual Pigment Optimizes Active State to Achieve Efficient G Protein Activation as Compared with Cone Visual Pigments

“…2 and Table 1). Our data indicated that V max /R*, V max /MII, and F Meta-II of photoactivated bvRh in nanodiscs were in good agreement with those of bvRh in ROSs (Tables 1 and 3), which is consistent with previous studies (18,35). Thus, these results demonstrated that nanodiscs mimic the native membrane environment and that the above mentioned molecular properties of visual pigments in the physiological environment can be studied using nanodisc samples.…”

“…Additionally, we also observed Meta-II formation (b1 and b2) of bvRh in ROSs and DM suspension at 20°C. At 20°C, the time constants of Meta-II formation for recombinant bvRh in POPC/POPG nanodiscs (6.23 and 47.7 ms) were similar to those of native bvRh in ROSs (8.65 and 37.8 ms), like those of native bvRh in nanodiscs (35). At this time scale, decay of Meta-II was not observed.…”

A Constitutively Activating Mutation Alters the Dynamics and Energetics of a Key Conformational Change in a Ligand-free G Protein-coupled Receptor

“…1A) on TM6 of opsin and on opsin containing a CAM, M257Y (24), and then compared their spectral properties to look for mutationlinked changes in the structure. Our results show that both in detergent micelles and in lipid conditions (using so-called nanodiscs, or NABBs) (25)(26)(27), opsin is constrained to an inactive conformation, whereas the CAM converts into a more active shape.…”

Conformational equilibria of light-activated rhodopsin in nanodiscs

“…In the present study, high-resolution SDSL-DEER distance mapping using pairs of spin labels is used to monitor the positions of TM5, TM6, and TM7 of rhodopsin in DDM and in phospholipid nanodiscs in which the receptor has properties similar to those in native membranes (26,27). Distance mapping with DEER overcomes the limitations in CW lineshape analysis and provides a length scale to the energy landscape.…”