The pH dependence of the rate constant of dark adaptation (thermal isomerization from all-trans- to 13-cis-bR) drastically changes when Arg82 of bacteriorhodopsin is replaced by an alanine. In the wild type (WT) the rate decreases sharply between pH 2.5 and pH 5. In R82A the sharp decrease is shifted to pH > 7. This correlates with the shift in the pK of the purple-to-blue transition from pH 2.6 in the wild type to pH 7.2 in the mutant (in 150 mM KCl). We propose that the same group that controls the purple-to-blue transition, namely, Asp85, catalyzes dark adaptation. The rate of dark adaptation in the R82A mutant is proportional to the fraction of protonated Asp85, indicating that dark adaptation occurs when Asp85 is transiently protonated. Thermal isomerization is at least 2 x 10(3) times more likely when Asp85 is protonated (blue membrane) than when it is deprotonated (purple membrane). The pH dependence of dark adaptation in the WT can be explained by a model in which the rate of dark adaptation in the WT is also proportional to the fraction of protonated Asp85 and that the pK of Asp85 depends on some other group, X, which deprotonates (or moves away from Asp85) with pK9 and causes the shift in the pK of Asp85 from 2.6 to 7.2. The quantum yield of light adaptation is at least an order of magnitude less in R82A as compared to the WT. The rise time of M formation is very fast in R82A and, unlike the WT, pH independent (1 microsecond versus 85 and 6 microseconds in the WT at pH 7 and 10, respectively). The activation energy of the L to M transition is 6.9 kcal/mol versus 13.5 kcal/mol in the WT. Thus the loss of a positive charge in the active site greatly increases the rate of light-induced deprotonation of the Schiff base. In the R82A mutant, the M decay at pH > 8.8 is much faster than the recovery of initial bR, which suggests a decrease in the rate of back-reaction from N to M. In a suspension of R82A membranes the rate of proton release as measured by the pH-sensitive dye pyranine is delayed by at least 20-fold (in 2 M KCl), while the uptake of protons did not change much (12 ms in the WT versus 8 ms in R82A).(ABSTRACT TRUNCATED AT 400 WORDS)

No abstract

The intramolecular interactions that stabilize the inactive conformation of rhodopsin are of primary importance in elucidating the mechanism of activation of this and other G protein-coupled receptors. In the present study, site-directed spin labeling is used to explore the role of a buried salt bridge between the protonated Schiff base at K296 in TM7 and its counterion at E113 in TM3. Spin-label sensors are placed at positions in the cytoplasmic surface of rhodopsin to monitor changes in the structure of the helix bundle caused by point mutations that disrupt the salt bridge. The single point mutations E113Q, G90D, and A292E, which were previously reported to cause constitutive activation of the apoprotein opsin, are found to cause profound movements of both TM3 and TM6 in the dark state, the latter of which is similar to that caused by light activation. The mutant M257Y, which constitutively activates opsin but does not disrupt the salt bridge, is shown to cause related but distinguishable structural changes. The double mutants E113Q͞M257Y and G90D͞M257Y produce strong activation of the receptor in the dark state. In the E113Q͞M257Y mutant investigated with spin labeling, the movement of TM6 and other changes are exaggerated relative to either E113Q or M257Y alone. Collectively, the results provide structural evidence that the salt bridge is a key constraint maintaining the resting state of the receptor, and that the disruption of the salt bridge is the cause, rather than a consequence, of the TM6 motion that occurs upon activation.G protein-coupled receptor ͉ signal transduction ͉ EPR ͉ site-directed spin labeling R hodopsin, the vertebrate dim-light photoreceptor, is the prototypic and best-studied member of the largest known superfamily of cell surface receptors, the G protein-coupled receptors. These receptors all contain seven transmembrane helical segments (TM1-TM7), as was clearly established in early structural studies of rhodopsin by cryoelectron microscopy (1) and confirmed more recently at higher resolution in 3D x-ray crystal structures of the dark state of the protein (2-4). In addition to the seven transmembrane helices, the x-ray structures reveal a short eighth helix in the C-terminal segment of the protein lying along the cytoplasmic surface of the membrane (H8, Fig. 1 A and B).The chromophore in rhodopsin, 11-cis-retinal, is bound to the protein covalently by means of a protonated Schiff linkage to the -amino group of Lys-296 located in TM7. The buried positive charge on the Schiff base nitrogen is stabilized by an electrostatic interaction, a salt bridge, with the charged carboxylate of Glu-113 in TM3 (Fig. 1C) (5-7). Capture of a photon by rhodopsin results in isomerization of retinal to the all-trans form. The isomerization triggers a series of transient conformational changes in the protein culminating in the formation of metarhodopsin II (MII), the active conformation (8). Concomitant with the formation of MII, the Schiff base nitrogen is deprotonated (9) and the Glu-113͞Lys-296 salt bri...