The role of a specific serine located in the retinylidene chromophore-binding pocket of bovine rhodopsin was investigated to determine its role in the mechanism of receptor photoactivaiton. The S186A mutant of rhodopsin was expressed in HEK293S GnTIcells, and the UV-vis absorbance change of the pigment in n-dodecylβ-D-maltoside detergent was measured as a function of time after photoexcitation with 7 ns laser pulses. Although S186A showed a normal bathorhodopsin (Batho), the microscopic rate constant for the back reaction of S186A blue-shifted intermediate (BSI) to Batho and the forward reaction of S186A BSI to lumirhodopsin (Lumi) were both significantly reduced. Furthermore, the UV-vis absorption maximum of S186A BSI was red-shifted by almost 20 nm relative to that of wild-type BSI, and the deprotonation of the Schiff base was unusually rapid and was complete in microseconds. The observed large mutagenic perturbations to the kinetics and photointermediate spectra suggest that the hydroxyl group of Ser186 interacts with the protonated Schiff base and/or its counterion after photoexcitation. A model is proposed for reorientation of the hydroxyl group of Ser186 upon formation of BSI that is part of a rearrangement of the hydrogen-bond network in the chromophore-binding pocket to facilitate the switch of counterion from Glu113 to Glu181 in the photoactivation process of rhodopsin.
Time-resolved absorbance difference spectra were collected at delays from 1 to 128 micros after photolysis of membrane and detergent suspensions of rhodopsin at 20 degrees C. Fitting both sets of data with two exponential decays plus a constant showed a similar fast process (lifetime 11 micros in membrane, 12 micros in 5% dodecyl maltoside) with a small but similar spectral change. This demonstrates that the Lumi I - Lumi II process, previously characterized in detergent suspensions, has similar properties in membrane without significant effect of detergent. The slower exponential process detected in the data is quite different in membrane compared to detergent solubilized samples, showing that the pronounced effect of detergent on the later rhodopsin photointermediates begins fairly abruptly near 20 micros. Besides affecting the late processes, the data collected here shows that detergent induces a small blue shift in the 1 micros difference spectrum (the Lumi I minus rhodopsin difference spectrum). The blue shift is similar to one induced by chloride ion in the E181Q rhodopsin mutant and may indicate that the ionization state of Glu181 in rhodopsin is affected by detergent.
Time-resolved absorbance measurements, over a spectral range from 300 to 700 nm, were made at delays from 1 μs to 2 ms after photoexcitation of bovine rhodopsin in hypotonically washed membrane suspensions over a range of temperature from 10 to 35 °C. The purpose was to better understand the reversibility of the Lumi I -Lumi II process that immediately precedes Schiff base deprotonation in the activation of rhodopsin under physiological conditions. To prevent artifacts due to rotation of rhodopsin and its photoproducts in the membrane, probe light in the timeresolved absorbance studies was polarized at the magic angle (54.7 degrees) relative to the excitation laser polarization axis. The difference spectrum associated with the Lumi I to Lumi II reaction was found to have larger amplitude at 10 °C compared to higher temperatures, suggesting that a significant back reaction exists for this process and that an equilibrated mixture forms. The equilibrium favors Lumi I entropically, and van't Hoff plot curvature shows the reaction enthalpy depends on temperature. The results suggest that Lumi II changes its interaction with the membrane in a temperature dependent way, possibly binding a membrane lipid more strongly at lower temperatures (compared to its precursor). To elucidate the origin of the time-resolved absorbance changes, linear dichroism measurements were also made at 20 °C. The time constant for protein rotation in the membrane was found to be identical to the time constant for the Lumi ILumi II process which is consistent with a common microscopic origin. We conclude that Lumi II (the last protonated Schiff base photointermediate under physiological conditions) is the first photointermediate whose properties depend on the protein-lipid environment.Considerable progress has been made during the last decade, since publication of the first 3-D crystal structure of rhodopsin (1), toward understanding the visual pigment activation mechanism, and more generally, that of G protein-coupled receptors (GPCRs). Beginning with simple photoisomerization of the 11-cis-N-retinylidene chromophore, rhodopsin activation threads through multiple substructures and protonation "switches" at diverse locations within the transmembrane region of the protein (2-4). Beyond those diverse motifs internal to the protein, it has long been known that rhodopsin's lipid environment powerfully affects the later stages of activation. However, causal relationships between all these parts and even the temporal sequence of their change have been difficult to establish, largely because experimental methods capable of resolving detailed structure require preparations stabilized using non-physiological conditions. In order to better establish the sequence of events in visual pigment activation so that they can be understood in vivo, time-resolved measurements under physiological conditions are essential.A primary advantage of time-resolved absorbance measurements reported here, is their ability to characterize equilibria which have been sh...
Bovine rhodopsin photointermediates formed in 2D rhodopsin crystal suspensions were studied by measuring the time dependent absorbance changes produced after excitation with 7 nanosecond laser pulses at 15, 25 and 35 °C. The crystalline environment favored the Meta I 480 photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspensions at 35 °C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the 380 nm absorbing product of Meta I 480 decay did not display the anomalous pH dependence characteristic of classical Meta II in the native disk membrane. Crystal suspensions bleached at 35°C and quenched to 19 °C showed that a rapid equilibrium existed on the ∼1 second time scale which suggests that the unprotonated predecessor of Meta II in the native membrane environment (sometimes called MII a ), forms in 2D rhodopsin crystals, but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and does not result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data of the 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changes and the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in the native membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach due to the random orientation of linearly dichroic crystals relative to the linearly polarized laser was explored as a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nm product of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activation suggests that determination of its structure would be of interest.Rhodopsin, the visual pigment used in dim light, originally attracted attention as the majority photoreceptor protein in mammalian retinas, with study of the intermediates in its photoactivation sequence starting long before its function as a G protein-coupled receptor (GPCR) was revealed (1). In virtually all other GPCRs the study of activation intermediates is extremely difficult because the diffusional nature of chemoreception does not allow the rapid triggering required for early intermediate characterization using ensemble based methods. While progress has been made in detecting intermediates in chemoreceptor GPCRs (2-4), it is only with great effort that processes 10,000 times slower than those reported here are studied. The detailed structural information available for the inactive, dark state of rhodopsin (5,6) provides an unambiguous basis for structural modeling of the earliest photointermediates. Optical methods of detection, such as time-resolved absorbance measurements (7), provide the highest time resolution of any experimen...
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