PMC

Activation mechanism of rhodopsin based on ligand-induced population shift. Meta-I, Meta-II, opsin, and possibly dark-state rhodopsin are in the equilibria between active and inactive conformations. Agonist all-trans retinal shifts the equilibrium toward active conformation by depressing ΔG, whereas inverse agonist 11-cis retinal shifts the equilibrium toward inactive conformation by elevating ΔG. Note that ΔG for Meta-I^DMPC would be greater than that for Meta-I in asolectin liposomes. To see this figure in color, go online.

Typical fluorescent spots of Rh/Alexa594 observed by TIRFM. (a) A typical TIRFM image. The images were obtained at 20 frames/s. The background was subtracted by a Rolling Ball Background Subtraction in ImageJ. (b and c) Analysis of fluorescence intensity of the fluorescent spot. The fluorescence intensity was estimated by averaging 12 × 12 pixels square containing a fluorescent spot. The average intensity of five 12 × 12 pixels squares in which no spot existed was subtracted as the background. Fluorescence intensity steps were detected by using a laboratory-written program based on a hidden Markov model (black line). See also .

Duration times for F_high and F_low in photoactivated rhodopsin. Histograms of duration time after fluorescence increase (F_high, blue squares) and decrease (F_low, red circles) at pH 6 (a), pH 7 (b), pH 8 (c), and pH 9 (d). Plots were fitted with a single-exponential function, and the residuals of the fit are shown above. Rate constants for conformational changes (k_low→high or k_high→low) were obtained according to Eqs. 4 and 5 (). (e) pH dependency of k_low→high (red) and k_high→low (blue). (f) pH dependency of the ratio of F_high (blue square) and Meta-II (red circle). The ratio of Meta-II was determined by UV-visible absorption measurements. Data were fitted with a Henderson-Hasselbalch equation . pK_a was 7.16 for Meta-I/Meta-II (red), and 6.98 for F_low/F_high (blue). To see this figure in color, go online.

Correlation between frequencies of conformational changes and G protein activation efficiencies. Intensity change histograms for dark-state (7m-Rh) (a), Meta-I in DMPC liposome (b), Meta-I/Meta-II mixture in asolectin liposomes (c), opsin (d), and denatured rhodopsin (e). These histograms were fitted with two mirror-imaged pairs of Gaussian curves (Eq. 2), where the parameters for noise components were global (μ₁ = 0.346, σ₁ = 0.113, A₁ = 0.012). Additional components were observed for Meta-I^DMPC (μ₂ = 0.201, σ₂ = 0.041, A₂ = 0.002), Meta-I/Meta-II (μ₂ = 0.171, σ₂ = 0.054, A₂ = 0.040), and opsin (μ₂ = 0.279, σ₂ = 0.072, A₂ = 0.005). (f) The frequency of fluorescence changes obtained by the summations of difference intensity histogram. Error bar indicates the standard deviation of two to five independent data sets. Frequency for the noise component is shown by shade. The asterisk indicates that the difference from denatured rhodopsin is significant (p < 0.05; Student’s t-test, two-tailed). (g) G protein activation efficiencies estimated by GTPγS binding assay. To see this figure in color, go online.

Histograms of fluorescence intensity of spots and difference intensity observed by TIRFM. (a) Histograms of maximum intensity in asolectin liposomes at pH 6 (n = 1850) fitted with three Gaussian functions shown in Eq. 1 (μ = 154, σ = 45, A₁ = 186, A₂ = 59, A₃ = 6). (b–d) Histograms of the fluorescence decrease upon photobleaching of Alexa594 in F_high (b), F_low (c), and unidentified state because of photobleaching before the fluorescence alternation (d). They were fitted with the single Gaussian functions [n = 676, μ = −160, σ = 51, A = 99 for (b), n = 471, μ = −126, σ = 46, A = 78 for (c), and n = 703, μ = −145, σ = 38, A = 134 for (d)]. (e–g) Difference intensity histograms for photoactivated rhodopsin (n = 924) (e), denatured rhodopsin (n = 505) (f), and free Alexa594 molecules in solution (n = 129) (g). The difference intensity histogram in (e) was well fitted with two mirror-imaged pairs of Gaussian functions (Eq. 2: μ₁ = ±0.304, σ₁ = 0.111, A₁ = 0.014, and μ₂ = ±0.167, σ₂ = 0.049, A₂ = 0.037), whereas those in (f) and (g) were fitted with a single mirror-imaged pair of Gaussian functions [μ = ±0.344, σ = 0.112, A = 0.011 (f), and μ = ±0.377, σ = 0.127, A = 0.011 (g)].