J.L. Bourdelande

Opening the Schiff base moiety of bacteriorhodopsin by mutation of the four extracellular Glu side chains

The quadruple bacteriorhodopsin (BR) mutant E9Q+E74Q+E194Q+E204Q shows a λ max of about 500 nm in water at neutral pH and a great influence of pH and salts on the visible absorption spectrum. Accessibility to the Schiff base is strongly increased, as detected by the rapid bleaching effect of hydroxylamine in the dark as well as in light. Both the proton release kinetics and the photocycle are altered, as indicated by a delayed proton release after proton uptake and changed M kinetics. Moreover, affinity of the color‐controlling cation(s) is found to be decreased. We suggest that the four Glu side chains are essential elements of the extracellular structure of BR.

Inclusion complex of calix [8] arene-C60 : photophysical properties and its behaviour as singlet molecular oxygen sensitiser in the solid state

Abstract The inclusion complex calix[8]arene-C 60 ) has been studied in the solid state by means of nanosecond laser flash photolysis with diffuse reflectance (DRLFP) and NIR luminescence (TRNIR) detection techniques. The recorded transient absorption spectrum and its kinetics are similar to those showed by the inclusion complex of γ -cyclodextrin-C 60 in solid state. Contrarily, the new complex shows a very different behaviour against oxygen: the deduced deactivation quenching constant by molecular O 2 is k q (O 2 )=(1.1 ±0.1) × 10 8 dm 3 mol −1 s −1 in solid state, while the deactivation of triplet γ -cyclodextrin-C 60 by molecular O 2 in solid state had been reported as negligible. Consistent with this, TRNIR shows unequivocally that the solid calix[8]arene complex photosensitises the formation of singlet molecular oxygen, O 2 ( 1 † g ). while the γ -cyclodextrin complex does not. O 2 ( 1 † g ) has a lifetime of 16 μs in the heterogeneous solid calix[8]arene complex-air system. Both complexes seem to show complementary properties to be used in the area of new materials.

The role of proline residues in the dynamics of transmembrane helices: the case of bacteriorhodopsin.

Proline residues in transmembrane helices have been found to have important roles in the functioning of membrane proteins. Moreover, Pro residues occur with high frequency in transmembrane α-helices, as compared to α-helices for soluble proteins. Here, we report several properties of the bacteriorhodopsin mutants P50A (helix B), P91A (helix C) and P186A (helix F). Compared to wild type, strongly perturbed behaviour has been found for these mutants. In the resting state, increased hydroxylamine accessibility and altered Asp-85 pKa and light-dark adaptation were observed. On light activation, hydroxylamine accessibility was increased and proton transport activity, M formation kinetics and FTIR difference spectra of M and N intermediates showed clear distortions. On the basis of these alterations and the near identity of the crystalline structures of mutants with that of wild type, we conclude that the transmembrane proline residues of bacteriorhodopsin fulfil a dynamic role in both the resting and the light-activated states. Our results are consistent with the notion that mutation of Pro to Ala allows the helix to increase its flexibility towards the direction originally hindered by the steric clash between the ring Cγ and the carbonyl O of the i-4 residue, at the same time decreasing the mobility towards the opposite direction. Due to their properties, transmembrane Pro residues may serve as transmission elements of conformational changes during the transport process. We propose that these concepts can be extended to other transmembrane proteins.

HIGHLY DIASTEREOSELECTIVE 2+2 PHOTOCYCLOADDITION OF HOMOCHIRAL 2(5H)-FURANONES TO VINYLENE CARBONATE

Abstract The [2+2] photocycloaddition of homochiral 5-alkyl-2(5 H )-furanones to vinylene carbonate was studied in order to enhance the induced facial diastereoselectivity created by the stereogenic centre. Diastereomeric excesses of up to 92% were found.

Inclusion complex of γ-cyclodextrin-C60: photophysical properties in the solid state using diffuse reflectance laser flash photolysis (DRLFP)

Abstract The transient behaviour of the solid γ-cyclodextrin-C60 complex was recorded by the diffuse reflectance laser flash photolysis (DRLFP) technique. The transient spectrum and kinetics of triplet decay are similar to those of the complex in aqueous solution, but there is a relevant absence of both triplet-triplet annihilation and molecular oxygen quenching processes.

Photocycloaddition of cyclohexene and maleic anhydride sensitized by insoluble benzoylated polystyrene

Abstract Friedel-Crafts benzoylation of styrene - 2% DVB copolymer beads yielded insoluble benzoylated polystyrene which was successfully used as sensitizer in the |2+2| photocycloaddition of cyclohexene with maleic anhydride.

Facile and stereoselective synthesis of fused γ-butyrolactones of carbohydrates

Abstract Photocycloadducts obtained from different homochiral butenolides and vinylene carbonate are shown to be useful precursors of fused γ-butyrolactones of carbohydrates.

Tris (bipyridyl) ruthenium(II) complex covalently bound to an insoluble hydrophilic polymer: reductive and oxidative quenching

Abstract The synthesis of a new insoluble, hydrophilic, polymeric equivalent of the Ru(bpy) 3 2+ complex is described. Sephadex is used as a hydrophilic polymeric skeleton. Its photophysical properties are similar to those of Ru(bpy) 3 2+ in solution. The deactivation of this polymeric excited complex by methyl viologen (oxidative quenching) and N , N -dimethylaniline (reductive quenching) is very efficient in polar solvents, making it both a powerful oxidant and reductant species in photochemical reactions accomplished in heterogeneous phase.

Fullerenes bound to an insoluble polymeric phase: surface photophysical studies using diffuse reflectance laser flash photolysis (DRLFP)

Abstract Fullerenes C 60 and C 70 were covalently bound to an insoluble cross-linked (2% divinylbenzene (DVB)) polystyrene. The transient absorption of the polymeric C 60 triplet was recorded by diffuse reflectance laser flash photolysis (DRLFP) showing a lifetime similar to that described for the C 60 triplet dissolved in toluene. The deactivation by molecular OZ has a quenching constant two orders of magnitude smaller than in solution.

Cross‐Linking of Transmembrane Helices Reveals a Rigid‐Body Mechanism in Bacteriorhodopsin Transport

In membrane proteins, the mechanisms driving transmembrane (TM) helices through the conformational changes needed to accomplish the translocation of substrates or for intraprotein signal transduction are still a matter of debate. Do the helices follow an alternating mechanical rocker-switch mechanism or do they act as soft structures, which would allow a Brownian ratchet mechanism? Are the interruptions or the hinges in the middle of transmembrane helices necessary to allow individual movements of parts of the helices? The activation of the G-protein-coupled receptor (GPCR) subfamily of 7TM helical receptors seems to imply that transmembrane helices VI and VII move apart (cytoplasmic side) or together (extracellular side) following a global toggle-switch mechanism. This mechanism also relies on see-saw movements around proline bends to allow the independent movement of different halves of the TM helices. Bacteriorhodopsin (bR), as a structural homologue of 7TM GPCRs, is a suitable model to further explore these questions. In bR, conformational changes triggered by the absorption of a photon by the retinal chromophore are responsible for the active transport of a proton, through a series of intermediate transport steps named K, L, M, N, and O and known as the photocycle. Although the first half of the photocycle (K–M) involves minor and subtle structural changes, the major conformational changes happen in the second half, in which the proton-transfer events swap from the extracellular to the cytoplasmic domain. It has been proposed that an outward rotation around the conserved Pro186 of helix F, accompanied by a tilt of helix G, is required for water entry during the M–N intermediate transition. It is believed that this would facilitate the reprotonation of both the Schiff base and Asp96. Conformational changes of the E–F loop during the photocycle have also been described. X-ray diffraction, spin labeling, and fluorescence experiments have provided experimental details of such movements, although the functional relevance of these changes has been questioned. An attractive approach to get insight into the nature of these movements is to restrict the conformational changes occurring between the cytoplasmic ends of helices F and G in the protein. With this purpose, we introduced two cysteines at the strategic positions 166 (end of helix F) and 228 (end of helix G) and induced a disulfide bond in the double mutant E166C/A228C (Figure 1 and the Materials and Methods