Structural changes in the N and N' states of the bacteriorhodopsin photocycle

Abstract

The bacteriorhodopsin transport cycle includes protonation of the retinal Schiff base by Asp96 (M-->N reaction) and reprotonation of Asp96 from the cytoplasmic surface (N-->N' reaction). We measured distance changes between pairs of spin-labeled structural elements of interest, and in general observed larger overall structural changes in the N state compared with the N' state. The distance between the C-D loop and E-F interhelical loops in A103R1/M163R1 increased approximately 6 A in the N state and approximately 3 A in the N' state. The opposite trend of distance changes in V101R1/A168R1 and L100R1/T170R1 supports counterclockwise rotation of helix F in the N but not the N' state. Small distance increases were observed in S169R1/S226R1, but little change was seen in G106R1/G155R1. Taking earlier published EPR data into account, we suggest that structural changes of the E-F loop occur first, and then helices F and G begin to move together in the late M state. These motions then reach their maximum amplitude in the N state, evidently to facilitate the release of a proton from Asp96 and the formation of a proton-conduction pathway from Asp96 to the Schiff base. The structural changes reverse their directions and decay in the N' state.

Figure 1

Bacteriorhodopsin model (PDB code 1C3W) viewed from the cytoplasmic side. Side chains replaced by cysteine and modified by spin labels are shown as balls at the coordinates of the respective CB atoms. Pairs of balls in the same color show double cysteine mutants to which two spin labels are attached. The three white arrows represent the possible motional reorientation of helices F and G, and the E-F loop (at Met¹⁶³). The two dashed-line arrows show the possible counterclockwise rotation of helix F.

Figure 2

(a) Absorption spectra of V49A/S169R1/S226R1 without (gray) and during (black) illumination in pH 6.0 buffer (10 mM Pi/Na, 100 mM NaCl, 15% (w/w) glycerol). The decreased absorption in the photostationary state indicates accumulation of the N state. (b) The characteristic N − BR spectrum (gray) obtained by subtracting black spectra from gray spectra in panel a. This difference spectrum was fitted with a standard N − BR spectrum (black) (26) to calculate occupancy of the N intermediate. The black arrow shows 635 nm light from the laser scattered by the membranes.

Figure 3

EPR spectra without and during illumination, at pH 6 and 9, and 120 K. Panels a and b are A103R1/M163R1 at pH 6 and pH 9; c and d are V101R1/A168R1 at pH 6 and pH 9; e and f are L100R1/T170R1 at pH 6 and pH 9; g and h are S169R1/S226R1 at pH 6 and pH 9; and i and j are G106R1/G155R1 at pH 6 and pH 9, respectively. Gray, solid lines are the observed spin-interacting spectra of nonilluminated, doubly labeled mutants. Black, dashed lines are the observed spin-interacting spectra of illuminated samples. Black, solid lines are the expected noninteracting spectra, measured with the spin dilution method. For each mutant, the spectra are superimposed and normalized to the same spin number.

Figure 4

Distance distribution of V49A/A103R1/M163R1 calculated from the EPR spectra in Fig. 3, a and b. Panels a and b are distributions in the BR and N′ states, respectively, at pH 6; c and d are distributions in the BR and N states, respectively, at pH 9. The distance distribution was obtained by deconvoluting the spectra attributed to N or N′ after subtracting a scaled amount of the nonilluminated spectrum from the illuminated spectrum, according to the occupancy of N or N′ in Table 1. The vertical (normalized population) axes of the distributions are arbitrary and selected for convenience of display.

Figure 5

EPR difference spectra at 120 K, calculated by subtracting the nonilluminated EPR spectra from the illuminated spectra in Fig. 3. Panels a and b are A103R1/M163R1 at pH 6 and pH 9; c and d are V101R1/A168R1 at pH 6 and pH 9; e and f are L100R1/T170R1 at pH 6 and pH 9; g and h are S169R1/S226R1 at pH 6 and pH 9; and i and j are G106R1/G155R1 at pH 6 and pH 9, respectively. Black lines are the difference spectra, and gray shadows are the standard errors. Statistics were calculated from three sets of individual experiments for each nonilluminated and illuminated state. Spectra c–j were scaled up to a and b by multiplying by 3. The vertical axes are arbitrary.