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Figure 2. Properties of BR-WT stationary and transient photocurrents.Photocurrents of BR-WT expressed in Xenopus oocytes are shown, which were evoked either by continuous illumination with green light (A) and/or blue laser flashes (C,D,G). (A) BR-WT photocurrents induced by illumination with green light (grey bar) at 0 mV (red) and â100 mV (blue). (B) Current-voltage plots of normalized stationary photocurrents of BR-WT evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed line is simply drawn to guide the eye. (C, D, G) Green light-induced stationary and blue laser flash-induced transient currents of BR-WT recorded at 0 mV (C), â30 mV (D) and â100 mV (G), green light illumination is indicated by a grey bar above the current traces. According to the illumination scheme above panel (C), the signals shown in (C,D,G) are superpositions of 12 recordings, from which the first is drawn in red color. In each sweep of the protocol, two blue laser flashes (indicated as #1, #1â²â¦ #12, #12â² for the 12 traces) were applied: The first blue flash was given at time Îtâ=â25 ms after the start of illumination with green light, the second at Îtâ=â25 ms after illumination stop. From sweep to sweep, Ît increased by 25 ms up to 300 ms. (E) and (F) show the transient currents in response to blue laser flash #1 (trace1) and #12 (trace 12) during illumination with green light from panel (C) in higher magnification.
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Figure 3. Photocurrents of mutants BR-F171C and BR-F219L.Photocurrents of BR-F171C (A) and BR-F219L (E) induced by illumination with green light (grey bar) at 0 mV (red) and â100 mV (blue). (B,F) Current-voltage plots of normalized stationary photocurrents of BR-F171C (B) and BR-F219L (F) evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed lines connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C,D,G,H) Green light-induced stationary and blue laser flash-induced transient currents of BR-F171C at 0 mV (C) and â100 mV (D), and BR-F219L at 0 mV (G) and â100 mV (H). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Îtâ=â100 ms after start and the second at Îtâ=â100 ms after the end of illumination with green light. From sweep to sweep, Ît increased by 100 ms up to 1200 ms.
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Figure 4. Photocurrents of mutants BR-D96N and BR-D96G.Photocurrents of BR-D96N (A) and BR-D96G (F) induced by illumination with green light (grey bar) at 0 mV (red) and â100 mV (blue). (B,G) Current-voltage plots of normalized stationary photocurrents of BR-D96N (B) and BR-D96G (G) evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed lines connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C,H) Photocurrents of BR-D96N (C, same cell as in panel A) and BR-D96G (H, same cell as in panel F) after addition of 50 mM azide. (D,E,J,K) Green light-induced stationary and blue laser flash-induced transient currents of BR-D96N at 0 mV (D) and â100 mV (E), and of BR-D96G at 0 mV (J) and â100 mV (K). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Îtâ=â100 ms after start and the second at Îtâ=â100 ms after the end of illumination with green light. From sweep to sweep, Ît increased by 100 ms up to 1200 ms.
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Figure 5. Photocurrents of mutant BR-D96G/F171C/F219L (BR-tri).(A) Photocurrents of BR-tri induced by illumination with green light (grey bar) at 0 mV (red) and â100 mV (blue). (B) Current-voltage plot of normalized stationary photocurrents evoked by continuous green light. For each cell, the stationary current amplitude at 0 mV was used for normalization. The dashed line connecting the data points are drawn to guide the eye; for comparison, the corresponding WT curve from Fig. 2B is included as dotted line. (C) Photocurrents of BR-tri (same cell as in panel A) after addition of 50 mM azide. (D,G) Green light-induced stationary and blue laser flash-induced transient currents of BR-tri at 0 mV (D) and â100 mV (G). Green light illumination is indicated by grey bars. The shown signals are superpositions of 12 recordings according to the illumination protocol from Fig. 2C. In each sweep, the first blue flash was given at Îtâ=â100 ms after start and the second at Îtâ=â100 ms after the end of illumination with green light. From sweep to sweep, Ît increased by 100 ms up to 1200 ms. (E,F) Transient photocurrents of BR-tri (E) and BR-WT (F) in response to blue laser flashes in higher magnification. Blue laser flashes were either applied during continuous illumination with green light (left signals in E,F) or without illumination in the dark (right signals in E,F).
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Figure 6. Comparison of characteristic properties of photocurrent signals.Photocurrents in response to continuous green light are characterized by five parameters: the amplitude of the transient current peak at the beginning of illumination (a), a stationary current amplitude (b), an initial amplitude of the slow phase of current decay after the end of illumination (c), a relaxation time Ï1 for the decrease from the initial peak current to the stationary level and Ï3 for the slow current decay after the end of illumination. The time constants for the initial current increase (Ï0) at the beginning and for the initial current decrease after light switch-off (Ï2) are not resolved due to the limited time resolution in TEVC experiments. In each panel, the parameters for the shown signals are included for comparison.
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Figure 7. Structural details during the BR photocycle.Three-dimensional ball and stick representations of the retinal chromophore (yellow), coupled via a Schiff base to Lys-216 (nitrogen atom in red) are shown and distances from the Schiff base nitrogen to the carboxyl oxygens (black) of Asp-85 are indicated by green dashed lines according to the following structural coordinates: (A) BR ground state structure (PDB structure entry 1C3W) [9], (B) BR ground state structure (PDB structure entry 1FBB) [41], (C) structure of the M intermediate (PDB structure entry 1C3W) [9], (D) Ground state structure of the triple mutant BR-D96G/F171C/F219L (PDB structure entry 1FBK) [11].
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Figure 1. 3D structure of BR.Cartoon representation of the 3D structure of bacteriorhodopsin according to the coordinates in PDB structure entry 1C3W by Luecke et al. (1999) prepared with PyMol 1.0 software. The retinal chromophore (magenta) is covalently linked via a Schiff base to Lys-216 in helix G (orange), which - together with the primary proton acceptor (Asp-85) and proton donor group (Asp-96) - is depicted in ball-and-chain representation (oxygen atoms: red, carbon atoms: green, nitrogen atoms: blue). Also shown are Phe-171 and Phe-219, which, together with Asp-96, were mutated herein.
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