Leticia U. Colmenares

ANALYZING THE RED‐SHIFT CHARACTERISTICS OF AZULENIC, NAPHTHYL, OTHER RING‐FUSED AND RETINYL PIGMENT ANALOGS OF BACTERIORHODOPSIN*

Prompted by the near infrared‐absorbing properties of some of the azulenic bacteriorhodopsin (bR) analogs, we have analyzed their absorption characteristics along with 11 new related ring‐fused analogs and the corresponding Schiff bases (SB) and protonated Schiff bases (PSB). The following three factors are believed to contribute to the total red shift of each of the pigment analogs (αRS): perturbation of the basic chromophore (SB shift, ΔSB), protonation of the SB (PSB shift, PSBS) and protein perturbation (the opsin shift, OS). For each factor, effects of structural modifications were examined. For the red‐shifted pigments, percent OS has been suggested as an alternate way of measuring protein perturbation. Computer‐simulated chromophores provided evidence against any explanation involving altered shapes of the binding pocket as a major cause for absorption differences. Implications of the current bR results on preparation of further red‐shifted bR and possible application to visual pigment analogs are discussed.

The molecular basis for the high photosensitivity of rhodopsin.

Based on structural information derived from the F NMR data of labeled rhodopsins, rhodopsin crystal structure, and excited-state properties of model polyenes, we propose a molecular mechanism that accounts specifically for the causes of the well-known enhanced photoreactivity of rhodopsin (increased rates and quantum yield of isomerization). It involves the key features of close proximity of C-187 to H-12 and chromophore bond lengthening upon light absorption. The resultant “sudden punch” to H-12 triggers dual processes of decay of the Franck–Condon-excited rhodopsin, a productive directed photoisomerization and a nonproductive decay returning to the ground state as two separate molecular pathways [based on real-time fluorescence results of Chosrowjan, H., Mataga, N., Shibata, Y., Tachibanaki, S., Kandori, H., Shichida, Y., Okada, T. & Kouyama, T. (1998) J. Am. Chem. Soc. 120, 9706–9707]. The two processes are controlled by the local protein structure: an empty space provided by the intradiscal loop connecting transmembrane helices 4 and 5 and a protein wall composed of amino acid units in transmembrane 3. Suggestions, involving retinal analogs and rhodopsin mutants, to improve the unusually high photosensitivity of rhodopsin are proposed.

Probing for the Threshold Energy for Visual Transduction: Red‐Shifted Visual Pigment Analogs from 3‐Methoxy‐3‐Dehydroretinal and Related Compounds

Abstract— While azulenic retinal analogs failed to yield a red‐shifted visual pigment analog, the 9‐cis isomers of the push‐pull polyenals 3‐methoxy‐3‐dehydroretinal and 14F‐3‐me‐thoxy‐3‐dehydroretinal yielded iodopsin pigment analogs with absorption maxima at, respectively, 663 and 720 nm. The former gave a relatively stable batho product (700 nm) and was able to activate transducin. A lower activity was observed for the latter. One possible explanation for the combined results is that the excitation energies of these red‐shifted pigments are approaching the threshold energy for visual transduction (although at this time we cannot rigorously exclude a role of the added F‐atom in reducing the transducin activity).

FLUORINATED PHENYLRHODOPSIN ANALOGS. BINDING SELECTIVITY, RESTRICTED ROTATION AND 19F-NMR STUDIES

Abstract Results from interactions of the 11-cis and 9-cis isomers of eleven fluorinated phenylretinal analogs, prepared from fluorinated benzaldehydes, with bovine opsin have been examined. Five of these (2′,6′-bis-CF3, 2′,4′,6′-tris-CF3, 2′-CF3-6′-F, 2′-CF3-7-methyl and 2′-CF3,6′-F,8-F) formed pigments in moderate to high yields, thus allowing recording of their 19F-NMR spectra which revealed inhibited conformational equilibration when protein bound. Possible causes for binding selectivity and fluorine chemical shifts are discussed.

Modeling rhodopsin, a member of G-protein coupled receptors, by computer graphics. Interpretation of chemical shifts of fluorinated rhodopsins

Abstract— An attempt has been made to construct a 3‐D model of rhodopsin, a member of G‐protein coupled receptors. Sequence homology of rhodopsin with the latter was a factor considered in the modeling procedure. The constructed model has been used to compare currently available specific protein/substrate interaction information, the shape of the binding cavity derived from shape of binding retinal isomers and analogs and challenged to explain recently available results from a series of fluorinated rhodopsins.

Nmr studies of fluorinated visual pigment analogs

The 19F-nmr chemical shift data of isomeric pigments (11-cis and 9-cis) of four vinyl fluororhodopsins and two trifluororhodopsins have been recorded. When compared with model protonated Schiff bases, a set of F-nmr opsin shift parameter (FOS) was obtained. The data revealed regiospecific protein perturbations on the F-resonances. They can be interpreted in terms of specific protein interactions such as the postulated second point charge and other polar interactions as well as the common hydrophobic protein perturbation.

9-CF3 and 13-CF3–β-carotene, canthaxanthin and related carotenoids. Synthesis, characterization and electrochemical data

Abstract The preparation and characterization of the following trifluoromethylated carotenoids are reported: 9-CF3–, 13-CF3–, 9,9′-bis-CF3–, 13,13′-bis-CF3–β-carotene, 9-CF3– and 13-CF3–canthaxanthin, 13′-CF3– and, 9′-CF3–4-oxo-β-carotene, 13′-CF3–adonirubin and 3-dehydro-13′-CF3–canthaxanthin. The CF3 group exhibits a strong cis-directing effect leading primarily to the cis isomer (near the CF3 group, i.e., all-E) in the synthetic mixtures. The minor all-trans isomer could be enriched by sensitized irradiation; however, they were found to be only marginally stable at room temperature. The CF3 group also exhibits a stabilizing effect towards oxidation reactions. Spectral data and oxidation potentials of these fluorinated carotenoids are reported.

9,11-DIcis-12-FLUORORHODOPSIN. CHROMOPHORE PROPERTIES FROM 19F-NMR STUDIES

Abstract— From a 19F‐NMR study of 9,11‐dicis‐12‐fluororhodopsin and its photobleached product, we concluded that the initially formed chromophore retained its configuration and the photoproduct corresponded to the two‐bond isomerized all‐trans. Upon standing, it slowly isomerized to the 9‐cis isomer. The method represents a direct, non‐destructive procedure for determining configuration purity of the pigment formed. Its unique fluorine opsin shift value is consistent with the expected different orientation of the fluoro‐substituent in a dicis pigment.

Spiral hexatrienes. The hindered cis isomer of mini-carotene-3 and hexakis-(2,2′,4,4′,6,6′-trifluoromethyl)stilbene

Abstract Steric crowding in the cis isomer of “Mini-3”, a chain shortened triene analog of β-carotene, and hexakis-(2,2′,4,4′,6,6′-trifluoromethyl)stilbene forces the polyene chromophores to adopt a spiral conformation. Some of the associated unusual spectroscopic properties (UV-VIS and NMR) of these compounds and a rare 1,7-H shift process are described.

Isomers of 10-fluoro-20-13C-retinal and the corresponding rhodopsin analogs

Abstract The doubly-labeled 10-fluoro-20- 13 C-retinal has been prepared. Five isomers have been isolated and two isomeric rhodopsin analogs prepared. The latter are intended for REDOR distance measurements.