Temperature dependence of the lumirhodopsin I-lumirhodopsin II equilibrium.

Time-resolved absorbance measurements, over a spectral range from 300 to 700 nm, were made at delays from 1 micros to 2 ms after photoexcitation of bovine rhodopsin in hypotonically washed membrane suspensions over a range of temperature from 10 to 35 degrees 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 time-resolved 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 degrees 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 degrees C. The time constant for protein rotation in the membrane was found to be identical to the time constant for the Lumi I-Lumi 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.

Rhodopsin photointermediates in two-dimensional crystals at physiological temperatures.

Bovine rhodopsin photointermediates formed in two-dimensional (2D) rhodopsin crystal suspensions were studied by measuring the time-dependent absorbance changes produced after excitation with 7 ns laser pulses at 15, 25, and 35 degrees 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 degrees 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 degrees C and quenched to 19 degrees C showed that a rapid equilibrium existed on the approximately 1 s 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.

Lumi I --> Lumi II: the last detergent independent process in rhodopsin photoexcitationt.

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.