Ingrid Wallat

Monitoring the conformational changes of photoactivated rhodopsin from microseconds to seconds by transient fluorescence spectroscopy.

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.

Location of chemically modified lysine 41 in the structure of bacteriorhodopsin by neutron diffraction.

Purple membranes were prepared in which bacteriorhodopsin was labeled at lysine 41 with phenylisothiocyanate (PITC) and with perdeuterated PITC. The in-plane position of this small label containing only five deuterons was determined from the differences between the neutron diffraction intensities of the two samples. At 8.7-A resolution the Fourier difference map revealed a well-defined site between helices 3 and 4. This position was confirmed by a refinement procedure in reciprocal space. Model calculations showed that the observed difference density had the right amplitude for the label. Thus it is possible to locate a small group in a large protein structure by replacing as few as five hydrogens by deuterium. The observed location of PITC restricts the number of possibilities for the assignment of helix B in the sequence (to which lysine 41 is attached) to one of the seven helices of the structure. Taking into account the size of the label and the length of the lysine side chain our result excludes helices 1, 2, and 7 as candidates for B.

Model Systems for the Investigation of the Opsin Shift in Bacteriorhodopsin

Donor-acceptor substituted styrenes and phenylbutadienes with substituents varying in donor and acceptor strength and as reconstituted chromophore-protein complexes were investigated as model compounds for the protonated Schiff base chromophore in bacteriorhodopsin (bR) both experimentally and theoretically. Charge distribution, donor-acceptor strength, and the shift of the absorption energy are correlated. The effect of the external electrostatic field was tested with a compound carrying an additional nonconjugated charge. The concept of overpolarization by the external charge, that is, the reversal of the relative importance of the two main resonance structures in S(0) and S(1), has been emphasized and related to a simple qualitative 2 x 2 interaction model. The variable donor approach is a new way for a better understanding of the Opsin shift in Bacteriorhodopsin.

Bacteriorhodopsin: the effect of bilayer thickness on 2D-array formation, and the structural re-alignment of retinal through the photocycle

From our earlier extensive protein-lipid reconstitution studies, the conditions under which bacteriorhodopsin forms organised 2D arrays in large unilamellar vesicles have been established using freeze-fracture electron microscopy. In a background bilayer matrix of phosphatidylcholine (diC(14:0)), the protein can form arrays only when the anionic purple membrane lipid, phosphatidylglycerol phosphate (or the sulphate derivative) is present. Here we have now extended this work to investigate the effect of bilayer thickness on array formation. Phosphatidylcholines with various chain lengths (diC(12:0), diC(14:0) and diC(16:0)) and which form bilayers of well defined bilayer thickness, have been used as the matrix into which bacteriorhodopsin, together with minimal levels (c. 4-10 lipids per bacteriorhodopsin) of diphytanyl phosphatidyl-glycerol phosphate, has been reconstituted. Arrays are formed in all complexes and bhickness appears only to alter the type of array formed, either as an orthogonal or as an hexagonal array. Secondly, we have previously deduced the entire conformation of retinal within the bacteriorhodopsin binding pocket in oriented purple membrane fragments. Using solid state deuterium NMR of the specifically deutero-methylated retinal labelled at each of the methyl positions in the molecule, the C-CD(3) bond vectors of the chromophore have been resolved to +/- 2 degrees . The ring conformation is 6-S-trans, but the polyene chain is slightly curved when in the protein binding site. Here, we describe studies on the protein in both the ground state and the trapped M(412)-state of the photocycle, to show that the orientation of the central methyl group (C(19)) on the polyene chain, which is at 40 degrees +/- 1 degrees with respect to the membrane normal, only changes its orientation by approximately 4 degrees upon 13-cis-isomerization. Thus, it is the Schiff base end of the chromophore which moves upon light incidence acting as a local switch on the protein in the photocycle, whilst the ring end of the chromophore moves rather less.