Protein-assisted pericyclic reactions: an alternate hypothesis for the action of quantal receptors. (65/2984)

The rules for allowable pericyclic reactions indicate that the photoisomerizations of retinals in rhodopsins can be formally analogous to thermally promoted Diels-Alder condensations of monoenes with retinols. With little change in the seven-transmembrane helical environment these latter reactions could mimic the retinal isomerization while providing highly sensitive chemical reception. In this way archaic progenitors of G-protein-coupled chemical quantal receptors such as those for pheromones might have been evolutionarily plagiarized from the photon quantal receptor, rhodopsin, or vice versa. We investigated whether the known structure of bacteriorhodopsin exhibited any similarity in its active site with those of the two known antibody catalysts of Diels-Alder reactions and that of the photoactive yellow protein. A remarkable three-dimensional motif of aromatic side chains emerged in all four proteins despite the drastic differences in backbone structure. Molecular orbital calculations supported the possibility of transient pericyclic reactions as part of the isomerization-signal transduction mechanisms in both bacteriorhodopsin and the photoactive yellow protein. It appears that reactions in all four of the proteins investigated may be biological analogs of the organic chemists' chiral auxiliary-aided Diels-Alder reactions. Thus the light receptor and the chemical receptor subfamilies of the heptahelical receptor family may have been unified at one time by underlying pericyclic chemistry.  (+info)

Time-resolved absorption and photothermal measurements with recombinant sensory rhodopsin II from Natronobacterium pharaonis. (66/2984)

Purified wild-type sensory rhodopsin II from Natronobacterium pharaonis (pSRII-WT) and its histidine-tagged analog (pSRII-His) were studied by laser-induced optoacoustic spectroscopy (LIOAS) and flash photolysis with optical detection. The samples were either dissolved in detergent or reconstituted into polar lipids from purple membrane (PML). The quantum yield for the formation of the long-lived state M(400) was determined as Phi(M) = 0.5 +/- 0.06 for both proteins. The structural volume change accompanying the production of K(510) as determined with LIOAS was DeltaV(R,1) /= Phi(M), indicating that the His tag does not influence this early step of the photocycle. The medium has no influence on DeltaV(R,1), which is the largest so far measured for a retinal protein in this time range (<10 ns). This confirms the occurrence of conformational movements in pSRII for this step, as previously suggested by Fourier transform infrared spectroscopy. On the contrary, the decay of K(510) is an expansion in the detergent-dissolved sample and a contraction in PML. Assuming an efficiency of 1.0, DeltaV(R,2) = -3 ml/mol for pSRII-WT and -4.6 ml/mol for pSRII-His were calculated in PML, indicative of a small structural difference between the two proteins. The energy content of K(510) is also affected by the tag. It is E(K) = (88 +/- 13) for pSRII-WT and (134 +/- 11) kJ/mol for pSRII-His. A slight difference in the activation parameters for K(510) decay confirms an influence of the C-terminal His on this step. At variance with DeltaV(R,1), the opposite sign of DeltaV(R,2) in detergent and PML suggests the occurrence of solvation effects on the decay of K(510), which are probably due to a different interaction of the active site with the two dissolving media.  (+info)

Magnesium binding to DM-nitrophen and its effect on the photorelease of calcium. (67/2984)

The effect of Mg(2+) on the process of Ca(2+) release from the caged Ca(2+) compound DM-nitrophen (NP) was studied in vitro by steady light UV photolysis of NP in the presence of Ca(2+) and Mg(2+). Ca(2+) release during photolysis and its relaxation/recovery after photolysis were monitored with the Ca(2+)-sensitive dye fura-2. Mg(2+) speeds the photorelease of Ca(2+) during photolysis and slows the relaxation of Ca(2+) to new steady-state levels after photolysis. Within the context of a model describing NP photolysis, we determined the on and off rates of Mg(2+) binding to unphotolyzed NP (k(on) = 6.0 x 10(4) M(-1) s(-1); k(off) = 1.5 x 10(-1) s(-1)). Furthermore, to fully account for the slow postphotolysis kinetics of Ca(2+) in the presence of Mg(2+) we were forced to add an additional photoproduct to the standard model of NP photolysis. The additional photoproduct is calculated to have a Ca(2+) affinity of 13.3 microM and is hypothesized to be produced by the photolysis of free or Mg(2+)-bound NP; photolysis of Ca(2+)-bound NP produces the previously documented 3 mM Ca(2+) affinity photoproduct.  (+info)

Blue- and green-absorbing visual pigments of Drosophila: ectopic expression and physiological characterization of the R8 photoreceptor cell-specific Rh5 and Rh6 rhodopsins. (68/2984)

Color discrimination requires the input of different photoreceptor cells that are sensitive to different wavelengths of light. The Drosophila visual system contains multiple classes of photoreceptor cells that differ in anatomical location, synaptic connections, and spectral sensitivity. The Rh5 and Rh6 opsins are expressed in nonoverlapping sets of R8 cells and are the only Drosophila visual pigments that remain uncharacterized. In this study, we ectopically expressed Rh5 and Rh6 in the major class of photoreceptor cells (R1-R6) and show them to be biologically active in their new environment. The expression of either Rh5 or Rh6 in "blind" ninaE(17) mutant flies, which lack the gene encoding the visual pigment of the R1-R6 cells, fully rescues the light response. Electrophysiological analysis showed that the maximal spectral sensitivity of the R1-R6 cells is shifted to 437 or 508 nm when Rh5 or Rh6, respectively, is expressed in these cells. These spectral sensitivities are in excellent agreement with intracellular recordings of the R8p and R8y cells measured in Calliphora and Musca. Spectrophotometric analyses of Rh5 and Rh6 in vivo by microspectrophotometry, and of detergent-extracted pigments in vitro, showed that Rh5 is reversibly photoconverted to a stable metarhodopsin (lambda(max) = 494 nm), whereas Rh6 appears to be photoconverted to a metarhodopsin (lambda(max) = 468 nm) that is less thermally stable. Phylogenetically, Rh5 belongs to a group of short-wavelength-absorbing invertebrate visual pigments, whereas Rh6 is related to a group of long-wavelength-absorbing pigments and is the first member of this class to be functionally characterized.  (+info)

Rapid-flow resonance Raman spectroscopy of photolabile molecules: rhodopsin and isorhodopsin. (69/2984)

We have devised a method for obtaining the resonance Raman spectrum of a photolabile molecule before it is modified by light. The essence of this technique is that the sample is flowed through the light beam at a sufficiently high velocity so that the fraction of photoisomerized (or photodestroyed) molecules in the illuminated volume is very low. This rapid-flow technique has enabled us to measure the resonance Raman spectrum of unphotolyzed bovine rhodopsin in Ammonyx LO detergent solution and in sonicated retinal disc membranes. The major features of these spectra, which are very similar to one another, are the protonated Schiff base line near 1660 cm-1, the ethylenic line at 1545 cm-1, lines due to skeletal modes at 1216, 1240, and 1270 cm-1, and a line due to C-H bending at 971 cm-1. The resonance Raman spectrum of unphotolyzed isorhodopsin formed by the addition of 9-cis-retinal to opsin was also measured. The spectrum of isorhodopsin is more complex and differs markedly from that of rhodopsin. In isorhodopsin, the ethylenic line is shifted to 1550 cm-1, and there are six lines between 1153 and 1318 cm-1. The rapid-flow technique described here makes it feasible to control the extent of interaction between light and any photolabile molecule. We present a theory for predicting the effective sample composition in the illuminated volume as a function of the flow rate, light intensity, and spectral characteristics of the photolabile species.  (+info)

Progress toward an explicit mechanistic model for the light-driven pump, bacteriorhodopsin. (70/2984)

Recent crystallographic information about the structure of bacteriorhodopsin and some of its photointermediates, together with a large amount of spectroscopic and mutational data, suggest a mechanistic model for how this protein couples light energy to the translocation of protons across the membrane. Now nearing completion, this detailed molecular model will describe the nature of the steric and electrostatic conflicts at the photoisomerized retinal, as well as the means by which it induces proton transfers in the two half-channels leading to the two membrane surfaces, thereby causing unidirectional, uphill transport.  (+info)

On the protein residues that control the yield and kinetics of O(630) in the photocycle of bacteriorhodopsin. (71/2984)

The effects of pH on the yield (phi(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O(630) intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and phi(r) at high pH [pK(a)(1) approximately 8]; 2) A rise in phi(r) at low pH [pK(a)(2) approximately 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) approximately 4. 5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi(r), k(r), and k(d) in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutant's data leads to the identification of the protein residue (R(1)) whose state of protonation controls the magnitude of phi(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O(630) parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R(2) and R(3). R(2) affects the rise of phi(r) at low pH, whereas the state of protonation of R(3) affects both k(r) and k(d). Our data confirm a previous suggestion that R(3) should be identified as the proton release moiety (XH). A clear identification of R(2), including its possible identity with R(3), remains open.  (+info)

Ovariectomy exacerbates and estrogen replacement attenuates photothrombotic focal ischemic brain injury in rats. (72/2984)

BACKGROUND AND PURPOSE: We previously reported the infarct volumes in female spontaneously hypertensive rats (SHR) to be significantly smaller than those in male SHR. The purpose of the present study was to determine whether estrogen is responsible for the sex difference in ischemic vulnerability in SHR. METHODS: In experiment 1, 1 week (short-term) or 4 weeks (long-term) after the ovariectomy (OVX), female SHR (5 months old) were randomly subjected to photothrombotic occlusion of the middle cerebral artery, and the infarct volumes were determined. In experiment 2, the rats were randomly assigned to 3 groups (ie, the sham-ovariectomized, ovariectomized, and estrogen replacement groups). In the replacement group, estradiol valerate (200 microgram/kg) was subcutaneously injected once a week after the OVX. Four weeks after the OVX or sham-OVX, all rats were subjected to middle cerebral artery occlusion. Changes in regional cerebral blood flow were determined by laser-Doppler flowmetry. RESULTS: In experiment 1, the infarct volume produced 1 week after the OVX was not different from that of the sham-ovariectomized group. In contrast, the infarct volume produced 4 weeks after the OVX was significantly larger than that of the sham-ovariectomized group (82.4+/-11.6 versus 54.5+/-16.0 mm(3), P=0.0058). In experiment 2, estradiol replacement after the OVX was observed to attenuate the infarct volume compared with the ovariectomized group (55.6+/-18.8 versus 78.5+/-21.0 mm(3), P=0.0321). The degrees of regional cerebral blood flow reduction did not differ among the sham-ovariectomized, ovariectomized, and estrogen replacement groups. CONCLUSIONS: Chronic estrogen depletion was thus found to increase the infarct size, which was attenuated by estradiol replacement. These findings indicate that estrogen contributes to the sex difference in ischemic vulnerability and that endogenous estrogen also has a neuroprotective effect against ischemic brain damage.  (+info)