1. Volume 21, Issue 3, Pages 426-437 (March 2013)
Kinetic, Energetic, and Mechanical Differences between Dark-State Rhodopsin and Opsin 
Shiho Kawamura, Moritz Gerstung, Alejandro T. Colozo, Jonne Helenius, Akiko Maeda, Niko Beerenwinkel, Paul S.-H. Park, Daniel J. Müller 
Structure 
Volume 21, Issue 3, Pages (March 2013)
DOI: /j.str
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2. Structure 2013 21, 426-437DOI: (10.1016/j.str.2013.01.011)
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3. Figure 1 Opsin Expressed in Rpe65−/− Mice Lacks 11-cis-Retinal
(A) UV/Vis absorbance spectroscopy (at 325 nm) of retinoids extracted from Rpe65−/− (solid line) or wild-type (dashed line) mice and separated by normal-phase high-performance liquid chromatography (HPLC). Samples from wild-type mice displayed absorbance peaks representing anti and syn isomers of 11-cis-retinal oximes (1, 1′) and anti and syn isomers of all-trans-retinal oximes (2, 2′). No retinals were observed in extracts from Rpe65−/− mice. To avoid overlap, traces for extracts from wild-type mice and Rpe65−/− mice were offset on the y axis. Other predominant peaks include the buffer exchange point (∗) and a nonretinoid peak (∗∗) with a maximum absorbance between 290−300 nm.
(B) UV/V is absorbance spectroscopy of opsin purified from Rpe65−/− mice (black line), opsin purified from Rpe65−/− mice, and reconstituted with 9-cis-retinal (gray line) and rhodopsin purified from wild-type mice (dashed line). Each experiment was repeated at least three times.
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4. Figure 2
F-D Curves Recorded upon Mechanically Unfolding Dark-State Rhodopsin or Opsin
(A and B) Individual F-D curves recorded during the mechanical unfolding of single rhodopsin (blue) and opsin (red) molecules. Force peaks represent the rupture of molecular interactions that stabilize unfolding intermediates of the receptor. F-D curves shown were recorded at a pulling velocity of 300 nm/s.
(C and D) Superimposed F-D curves of rhodopsin (n = 799) or opsin (n = 628) represented as density plots with the color spectrum (see color scale bar), indicating the frequency of force peaks. Rhodopsin data were taken from Kawamura et al. (2012).
See also Figures S1, S2, S4, and S5.
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5. Figure 3
Contour Length Histograms of Force Peaks Detected upon Unfolding of Dark-State Rhodopsin and Opsin
(A) Histograms of contour lengths at which force peaks were detected in F-D curves recorded from dark-state rhodopsin (blue) and opsin (red). Bin size, 1 amino acid (aa). A total of 799 and 628 F-D curves were analyzed for rhodopsin and opsin, respectively. The frequency difference, Δ, between rhodopsin and opsin is indicated below the histogram. Arrows indicate the contour length of force peak classes as determined by the Gaussian mixture model (B-C).
(B and C) Gaussian mixture model fits to determine the contour length and width of force peak classes in each histogram. In the rhodopsin histogram, ten force peak classes were determined. Each force peak class is represented by a weighted Gaussian model component and is indicated by a unique color. Color segments on the x axis indicate the range of contour lengths determined for each force peak class by the Bayes classifier. Boundaries of each force peak class are listed in Table 1. The same ten Gaussian model components fit the opsin histogram. The sums of weighted contour lengths detected for all force peaks, that is, the full mixture models, are shown as black lines. Gray bars denote histograms shown in (A). Dashed lines indicate the uniform baseline noise determined by Equation 2.
See also Figures S2–S5.
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6. Figure 4
Stable Structural Segments Detected in Dark-State Rhodopsin and Opsin
Secondary structure of rhodopsin and opsin with amino acid residues colored to highlight the stable structural segments ([N1], red), ([N2], blue), ([H1], purple), ([C1-H2], dark pink), ([E1], cyan), ([H3-C2-H4-E2], orange), ([H5-C3-H6.1], dark green), ([H6.2-E3-H7], yellow), ([H8], light pink), and ([CT], light green). Black numbers denote the amino acid residue at which a stable structural segment starts. Numbers in parentheses are contour lengths obtained by fitting contour length histograms (Table 1). Differences between values are due to the disulfide bridge (S-S) or membrane compensation (Experimental Procedures). The chromophore 11-cis-retinal forms a Schiff-base linkage at Lys296 (circled black).
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7. Figure 5 DFS Plots of Dark-State Rhodopsin and Opsin
Each DFS plot shows the dynamic behavior of a stable structural segment from dark-state rhodopsin (blue) and opsin (red). Plotted is the mean unfolding force of each stable structural segment versus loading rate. Slanted ellipses indicate one standard error of each data point. The Bell-Evans model was fitted (solid lines) to obtain the unfolding energy barrier parameters (Experimental Procedures; Equations S14 and S15). Dark- and light-colored regions indicate fitting confidence intervals of one (68%) and two (95%) standard deviations, respectively.
See also Figures S2, S6, and S7.
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8. Figure 6
Conformational, Kinetic, Energetic, and Mechanical Properties of Dark-State Rhodopsin and Opsin
(A and B) Structural models of (A) rhodopsin (Protein Data Bank [PDB] ID code 1U19) and (B) opsin (PDB ID code 3CAP) mapped with stable structural segments shown in Figure 4. The following structural models have been colored to map the distance separating the folded from the transition state, xu (e.g., conformational variability), the transition rate, k0 (reciprocal of lifetime), the unfolding free energy, ΔG, and the mechanical spring constant, κ. Color bars specify the range of values taken from Table 2. Maps were created using PyMOL Molecular Graphics System (v.1.2r3pre, Schrödinger).
See also Figure S7.
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9. Figure 7
Overall Conformational, Kinetic, Energetic, and Mechanical Differences between Dark-State Rhodopsin and Opsin
Compared to dark-state rhodopsin, the valleys of the energy barriers stabilizing structural segments of opsin are narrower (smaller xu). In addition, structural segments of opsin generally display a reduced lifetime (reciprocal of k0), are stabilized by a lower activation free energy (ΔG), and show increased mechanical rigidity (κ). Upon binding 11-cis-retinal, structural segments stabilizing rhodopsin increase structural flexibility, increase kinetic stability, and decrease mechanical rigidity.
Structure  , DOI: ( /j.str )
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