Photochemical nature of parietopsin

Abstract

Parietopsin is a nonvisual green light-sensitive opsin closely related to vertebrate visual opsins and was originally identified in lizard parietal eye photoreceptor cells. To obtain insight into the functional diversity of opsins, we investigated by UV-visible absorption spectroscopy the molecular properties of parietopsin and its mutants exogenously expressed in cultured cells and compared the properties to those of vertebrate and invertebrate visual opsins. Our mutational analysis revealed that the counterion in parietopsin is the glutamic acid (Glu) in the second extracellular loop, corresponding to Glu181 in bovine rhodopsin. This arrangement is characteristic of invertebrate rather than vertebrate visual opsins. The photosensitivity and the molar extinction coefficient of parietopsin were also lower than those of vertebrate visual opsins, features likewise characteristic of invertebrate visual opsins. On the other hand, irradiation of parietopsin yielded meta-I, meta-II, and meta-III intermediates after batho and lumi intermediates, similar to vertebrate visual opsins. The pH-dependent equilibrium profile between meta-I and meta-II intermediates was, however, similar to that between acid and alkaline metarhodopsins in invertebrate visual opsins. Thus, parietopsin behaves as an "evolutionary intermediate" between invertebrate and vertebrate visual opsins.

Figure 1

Schematic phylogenetic tree showing the relationship between vertebrate visual and non-visual opsins/encephalopsin group. Candidate residues for counterion (residues at 113 and 181 in the numbering system for bovine rhodopsin), and absorption maxima of each opsin groups are shown on the right.

Figure 2

pH-dependency of dark spectra of wild-type parietopsin and mutants. Wild-type lizard parietopsin (A), E181Q (B), Q113E/E181Q (C) and Q113E (D) were purified with CHAPS/PC buffer at pH 7.0 containing 20% (w/v) glycerol, and pH was varied by adding small amounts of HCl or NaOH. The numbers in graph traces indicate pH values. Absorption spectra were measured at 0 °C.

Figure 3

Photosensitivity of parietopsin and bovine rhodopsin. (A) Lizard parietopsin (curve 1) and (B) bovine rhodopsin (curve 1) were irradiated successively with 500-nm light at 0 °C and absorbance changes were monitored (curves 2-9). The samples were completely bleached at the end of the experiment (curves 10). (C) The amount of residual pigment was plotted against the incident photon number. The error bars represent the deviation of the two independent measurements. The photosensitivity of parietopsin relative to bovine rhodopsin was estimated from the slopes of the fitted lines. (D) Molar extinction coefficients (ε) of parietopsin and bovine rhodopsin. Bovine rhodopsin and parietopsin were denatured by adding HCl (final pH <1.5), which yielded identical denatured products (dashed lines). By normalizing the concentrations of parietopsin and bovine rhodopsin samples using the acid-denatured spectra, ε of parietopsin relative to bovine rhodopsin was calculated to be 31700 ± 500 M⁻¹cm⁻¹ at 520 nm and 29000 ± 500 M⁻¹cm⁻¹ at 500 nm using the value of 40600 M⁻¹cm⁻¹ at 500 nm for bovine rhodopsin.

Figure 4

Photochemical reaction of lizard parietopsin at −185 °C. Parietopsin in CHAPS/PC buffer with 91% (w/v) glycerol was cooled to −185 °C (curve 1), and irradiated with 500 nm light (< 1 mW/cm²) for 315 s to form a photo-steady-state mixture (curve 2). Then it was irradiated with deep-red light at >630 nm (11 mW/cm²) for 300 s (curve 3), followed by irradiation with 500-nm light for 300 s (curve 4).

Figure 5

Photobleaching process of lizard parietopsin. (A) Thermal reactions of bathoparietopsin. The photo-steady-state mixture mainly containing batho intermediate (curve 1) was warmed to −170 °C and recooled to −185 °C for the measurement of the spectrum (curve 2). Similarly the sample was warmed to −160, −150, −140, −130, −120, −110, −100 and −90 °C in a stepwise manner, and the spectrum was recorded at −185 °C after each warming (curves 3-10). (B) After the measurements shown in panel (A), the absorption spectrum of the sample was recorded at −80 °C (curve 1). It was warmed to −70, −60, −50, −40, −30, −20 and −10 °C in a stepwise manner, and the spectra were measured at −80 °C (curves 2-8). Inset: the difference spectra calculated by subtracting curve 1 from curves 6 and 8. (C) Transition from metaparietopsin-I to metaparietopsin-II at −20 °C. Parietopsin sample was irradiated with >560 nm light (20 mW/cm²) for 15 sec. The spectra were recorded at 1.5, 4.5, 9, 16, 32, 60, 120 and 150 min after irradiation (curves 1-8). Inset: the difference spectra calculated by subtracting curve 1. (D) Transition from metaparietopsin-II to metaparietopsin-III was observed at −10 °C. Parietopsin sample was irradiated with >560 nm light (20 mW/cm²) for 15 sec. The spectra were recorded at 1.5, 4.5, 9, 16, 32, 60, 120 and 150 min after irradiation (curves 1-8). Inset: the difference spectra calculated by subtracting curve 1. (E) Dissociation of metaparietopsin-II and metaparietopsin-III into retinal and opsin at 20 °C. Parietopsin sample was irradiated with >500 nm light (27 mW/cm²) for 20 sec. The spectra were recorded at 1.5, 5, 10, 15, 25, 60, 120 and 200 min after irradiation (curves 1-8). Inset: the difference spectra calculated by subtracting curve 1.

Figure 6

Photoreaction of detergent free parietopsin in membranes. (A) Transient absorption spectra of parietopsin in HEK293T membrane fragments at −20 °C. The sample was prepared by the sucrose flotation method and added 50% (w/v) glycerol. The spectra were recorded at 1.5, 5, 10, 30, 60 and 120 min after irradiation with >560 nm-light (20 mW/cm²) for 15 sec. Difference spectra was calculated by subtracting the spectrum recorded before irradiation from the spectra after irradiation (curves 1-6). Inset: the difference spectra between curve 1 (1.5 min) and curve 5 (60 min). (B) Transition from metaparietopsin-I to metaparietopsin-II plus metaparietopsin-III in PC liposomes. Parietopsin in PC liposomes (curve 1) was irradiated with a 532-nm laser pulse at 0 °C. The spectra recorded at 0.01, 0.05, 0.5, 1, 60, 600, 1800 sec after irradiation (curve 2-8) are displayed. (C) Difference spectra calculated by subtracting curve 2 from curves 3-8 shown in (B). Inset: Two b-spectra (BS1 and BS2) calculated from the spectral change shown in Figure 6C. BS1 and BS2 indicate transitions from metaparietopsin-I to metaparietopsin-II and from metaparietopsin-II to metaparietopsin-III, respectively. (D) Transition from metaparietopsin-II and metaparietopsin-III to retinal and apoprotein in PC liposomes. Parietopsin in PC liposomes (curve 1) was irradiated with >560 nm light (20 mW/cm²) for 5 min at 0 °C. The spectra were recorded at 5, 30 and 60 min and 2, 5, 10 and 14 hours after irradiation (curves 2-8). Inset: the difference spectra between curve 1 and curve 2, showing that metaparietopsin-II and metaparietopsin-III are formed by irradiation.

Figure 7

pH-equilibrium between metaparietopsin-I and metaparietopsin-II. The samples were purified with CHAPS/PC buffer containing 20% (w/v) glycerol. Transient spectra from metaparietopsin-I to metaparietopsin-II were recorded at 0 °C by using time-resolved CCD spectrophotometer. The difference spectrum at each pH was calculated by subtracting the spectrum measured at 10 ms after the flash irradiation from that of the equilibrium state measured at 100 sec. It should be noted that the spectrum measured at 10 ms after the irradiation is the composite of only two components, the spectra of metaparietopsin-I and unreacted parietopsin. The amount of unreacted parietopsin was determined by hydroxylamine bleaching of metaparietopsin-I after the time-resolved measurements, and subtracted from the spectra at 10ms to calculate the amount of metaparietopsin-I generated by the flash. In the main panel, difference spectra were normalized at their negative peaks (490 nm) relative to the amount of metaparietopsin-I produced by the irradiation. Inset: The ratio of metaparietopsin-I was estimated by the difference absorbance at 490 nm in the main panel. They were plotted against pH, and the curve was fitted by Henderson-Hasselbalch equation.