Showing papers by "Thorgeir E. Thorgeirsson published in 1991"

Effects of detergent environments on the photocycle of purified monomeric bacteriorhodopsin.

Abstract: Time-resolved difference spectra have been obtained for the photocycle of delipidated bacteriorhodopsin monomers (d-BR) in six different detergent micelle environments that were prepared by two new detergent-exchange techniques. A global kinetic analysis of the photocycle spectra for d-BR in each detergent environment was performed. Comparison of these results with those obtained for the photocycle of bacteriorhodopsin in purple membrane (PM) shows that there is one fewer kinetically distinguishable process for monomeric BR between the decay of the K intermediate and the rise of the M intermediate. Assuming a sequential pathway occurs in the photocycle, it appears that the equilibrium between the L and M intermediates is reached much more rapidly in the detergent micelles. This is attributed to a more direct interaction between Asp-85 and the proton on the nitrogen of the Schiff base of retinal for BR in the detergents. Equilibrium concentrations of late photocycle intermediates are also altered in detergents. The later steps of the photocycle, including the decay of the M intermediate, are slowed in detergents with rings in their hydrocarbon region. This is attributed to effects on conformational changes occurring during the decay of M and/or other later photocycle intermediates. The lifetime of dark adaptation of light-adapted d-BR in different detergent environments increases in environments where the lifetime of the M intermediate increases. These results suggest that the high percentage of either unsaturated or methyl-branched lipids in PM and the membranes of other retinal proteins may be important for their effective functioning.

Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin.

Abstract: Bacteriorhodopsin (BR) with the single-site substitutions Arg-82----Gln (R82Q), Asp-85----Asn (D85N), and Asp-96----Asn (D96N) is studied with time-resolved absorption spectroscopy in the time regime from nanoseconds to seconds. Time-resolved spectra are analyzed globally by using multiexponential fitting of the data at multiple wavelengths and times. The photocycle kinetics for BR purified from each mutant are determined for micellar solutions in two detergents, nonyl glucoside and CHAPSO, and are compared to results from studies on delipidated BR (d-BR) in the same detergents. D85N has a red-shifted ground-state absorption spectrum, and the formation of an M intermediate is not observed. R82Q undergoes a pH-dependent transition between a purple and a blue form with different pKa values in the two detergents. The blue form has a photocycle resembling that for D85N, while the purple form of R82Q forms an M intermediate that decays more rapidly than in d-BR. The purple form of R82Q does not light-adapt to the same extent as d-BR, and the spectral changes in the photocycle suggest that the light-adapted purple form of R82Q contains all-trans- and 13-cis-retinal in approximately equal proportions. These results are consistent with the suggestions of others for the roles of Arg-82 and Asp-85 in the photocycle of BR, but results for D96N suggest a more complex role for Asp-96 than previously suggested. In nonyl glucoside, the apparent decay of the M-intermediate is slower in D96N than in d-BR, and the M decay shows biphasic kinetics. However, the role of Asp-96 is not limited to the later steps of the photocycle. In D96N, the decay of the KL intermediate is accelerated, and the rise of the M intermediate has an additional slow phase not observed in the kinetics of d-BR. The results suggest that Asp-96 may play a role in regulating the structure of BR and how it changes during the photocycle.

Photolysis intermediates of human rhodopsin.

Abstract: Photochemical studies were conducted on human rhodopsin at 20 degrees C to characterize the intermediates which precede the formation of metarhodopsin II, the trigger for the enzyme cascade mechanism of visual transduction. Human rhodopsin was prepared from eyes which had previously been used for corneal donations. Time resolved absorption spectra collected from 10(-8) to 10(-6) s after photolysis of human rhodopsin in detergent suspensions displayed biexponential decay kinetics. The apparent lifetimes obtained from the data are 65 +/- 20 and 292 +/- 25 ns, almost a factor of 2 slower than the corresponding rates in bovine rhodopsin. The spectra can be fit well using a model in which human bathorhodopsin decays toward equilibrium with a blue-shifted intermediate (BSI) which then decays to lumirhodopsin. Spectra and kinetic rate constants were determined for all these intermediates using a global analysis which showed that the spectra of the human intermediates are remarkably similar to bovine intermediates. Microscopic rate constants derived from this model are 7.4 x 10(6) s-1 for bathorhodopsin decay and 7.5 x 10(6) s-1 and 4.6 x 10(6) s-1 for the forward and reverse reactions of BSI, respectively. Decay of lumirhodopsin to later intermediates was studied from 10(-6) to 10(-1) s after photolysis of rhodopsin in human disk membrane suspensions. The human metarhodopsin I in equilibrium metarhodopsin II equilibrium appears to be more forward shifted than in comparable bovine studies.

Mechanism of vibrational relaxation and intersystem crossing within excited ion-pair states of I 2

Abstract: Visible and ultraviolet fluorescence of I2, following excitation by ArF/193nm excimer laser pulses, was recorded for different pressures of argon buffer gas in a flow system. Dispersed fluorescence spectra due to the transitionsD’(2g) → A’(2y andD(0 n + )→X0 g + ) were analysed by inversion and spectral simulations. Thus vibrational distributions in the emitting states were obtained as a function of pressure to determine the mechanism of relaxation to populate the lowest quantum levels of theD’ state, which are the emitting states in the iodine laser. Fast intersystem crossing is found to occur from initially populated vibrational levels of theD state to other ion-pair states correlating with the ground state ions, followed by rapid relaxation, involving both direct vibrational relaxation within individual states and intersystem crossing between states.