Thomas G. Ebrey

Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment.

Previously we reported the sequence of the member of the short wavelength sensitive 2 (SWS2) family of vertebrate visual pigments from the retina of the Japanese common newt, Cynops pyrrhogaster[Takahashi, Y. et al. (2001) FEBS Lett. 501, 151-155]. Now we have expressed the apopigment and regenerated it with A1 retinal. Its absorption maximum, 474 nm, is greatly red shifted compared to other known SWS2 pigments (418-455 nm). To determine the amino acid residues that control its spectral tuning, we replaced the residues that were near the chromophore and which differed between the newt and the bullfrog (lambda(max) = 430 nm) wild-type SWS2 pigments: Pro91Ser, Ser94Ala, Ile122Met, Cys127Ser, Ser211Cys, Tyr261Phe, and Ala292Ser. Each of these site-directed mutants led to blue shifts of the newt pigment with five of them causing substantial shifts; their sum was about equal to the difference between the absorption maximum of the bullfrog and newt pigments, 44 nm. The 32 nm shift of the absorption maximum of the multiple seven-residue mutant to 442 nm is fairly close to that of the wild-type bullfrog pigment. Thus, the seven amino acid residues that we replaced are the major cause of the red shift of the newt SWS2 pigments spectrum. Two of the residues, 91 and 94, have not previously been identified as wavelength regulating sites in visual pigments. One of these, 91, probably regulates color via a new mechanism: altering of a hydrogen bonding network that is connected via a water to the chromophore, in this case its counterion, Glu113.

Kinetics and pH Dependence of Light-Induced Deprotonation of the Schiff Base of Rhodopsin: Possible Coupling to Proton Uptake and Formation of the Active Form of Meta II

In this paper we first review what is known about the kinetics of Meta II formation, the role and stoichiometry of protons in Meta II formation, the kinetics of the light-induced changes of proton concentration, and the site of proton uptake. We then go on to compare the processes that lead to the deprotonation of the Schiff base in bacteriorhodopsin with rhodopsin. We point out that the similarity of the signs of the light-induced electrical signals from the two kinds of oriented pigment molecules could be explained by bacteriorhodopsin releasing a proton from its extracellular side while rhodopsin taking up a proton on its cytoplasmic side. We then examined the pH dependence of both the absorption spectrum of the unphotolyzed state and the amplitude and kinetics of Meta II formation in bovine rhodopsin. We also measured the effect of deuteration and azide on Meta II formation. We concluded that the pKa of the counter-ion to the Schiff base of bovine rhodopsin and of a surface residue that takes up a proton upon photolysis are both less than 4 in the unphotolyzed state. The data on pH dependence of Meta II formation indicated that the mechanisms involved are more complicated than just two sequential, isospectral forms of Meta II in the bleaching sequence. Finally we examined the evidence that, like in bacteriorhodopsin, the protonation of the Schiff basess counter-ion (Glu113) is coupled to the changing of the pKa of a protonatable surface group, called Z for rhodopsin and tentatively assigned to Glu134. We conclude that there probably is such a coupling, leading to the formation of the active form of Meta II.

A purified agonist-activated G-protein coupled receptor: truncated octopus Acid Metarhodopsin.

Abstract G-protein coupled receptors (GPCRs) mediate responses to many types of extracellular signals. So far, bovine rhodopsin, the inactive form of a GPCR, is the only member of the family whose three dimensional structure has been determined. It would be desirable to determine the structure of the active form of a GPCR. In this paper, we report the large scale preparation of a stable, homogenous species, truncated octopus rhodopsin (t-rhodopsin) in which proteolysis has removed the proline-rich C-terminal; this species retains the spectral properties and the ability for light-induced G-protein activation of unproteolyzed octopus rhodopsin. Moreover, starting from this species we can prepare a pure, active form of pigment, octopus t-Acid Metarhodopsin which has an all-trans-retinal as its agonist. Photoisomerization of t-Acid Metarhodopsin leads back to the inactive form, t-rhodopsin with the inverse agonist 11-cis-retinal. Octopus t-Acid Metarhodopsin can activate an endogenous octopus G-protein in the dark and this activity is reduced by irradiation with orange light which photoregenerates t-Acid Metarhodopsin back to the initial species, t-rhodopsin.

Water as a cofactor in the unidirectional light-driven proton transfer steps in bacteriorhodopsin

Abstract Recent evidence for involvement of internal water molecules in the mechanism of bacteriorhodopsin is reviewed. Water O–H stretching vibration bands in the Fourier transform IR difference spectra of the L, M and N intermediates of bacteriorhodopsin were analyzed by photoreactions at cryogenic temperatures. A broad vibrational band in L was shown to be due to formation of a structure of water molecules connecting the Schiff base to the Thr46-Asp96 region. This structure disappears in the M intermediate, suggesting that it is involved in transient stabilization of the L intermediate prior to proton transfer from the Schiff base to Asp85. The interaction of the Schiff base with a water molecule is restored in the N intermediate. We propose that water is a critical mobile component of bacteriorhodopsin, forming organized structures in the transient intermediates during the photocycle and, to a large extent, determining the chemical behavior of these transient states.

Dynamic Holography in Bacteriorhodopsin/Gelatin Films: Effects of Light–Dark Adaptation at Different Humidity†

This work examines the kinetics of dynamic holography responses in light‐adapted and dark‐adapted bacteriorhodopsin (BR) films at different humidity. We have demonstrated that the kinetics of the diffraction efficiency in wild type BR films is quite different in dark‐adapted and light‐adapted samples. The holographic recording kinetics, which depends on the duration of incubation in the dark after light adaptation at different humidity values, was studied in depth. A specially designed miniature cell containing a BR film was mounted inside the holographic set up to allow controlled humidity changes over a broad range. The diffraction efficiency kinetics at humidity values of 96–99% were quite different from the kinetics at 60–93% humidity. We found that humidity values of 90–93% were most optimal for dynamic holography recording using a gelatin film containing BR. In agreement with a calculation of the wavelength‐dependent changes of the refractive index for dark‐adapted and light‐adapted BR samples using the Kramers–Kronig relation, the maximum difference in the refractive index and thus in the diffraction efficiency for dark‐adapted and light‐adapted BR films takes place at 630 nm, close to the wavelength of the He–Ne laser used.

Participation of Internal Water Molecules and Clusters in the Unidirectional Light-Induced Proton Transfer in Bacteriorhodopsin

Proton pumping is a fundamental biological process for generating free energy in the form of a transmembrane electrochemical potential for hydrogen ions; this energy is then utilized in cellular processes such as ATP synthesis, rotation of a flagellar motor, and ion transport across the plasma membrane. In the photosynthetic reaction center, light is used to induce charge separation and generate reducing and oxidizing equivalents.The subsequent electron flow through a chain of electron transporting proteins is coupled to the proton pumping, and so the pump makes indirect use of the light energy. In contrast a single small (26kDa) protein, bacteriorhodopsin (BR), not only absorbs the light energy and does the photochemistry, it also is the proton pump. Recently similar pigments have been found in many marine eubacteria where they provide a significant portion of the photosynthetic yield of the oceans.

Brighter than the sun: Rajni Govindjee at 80 and her fifty years in photobiology

We celebrate distinguished photobiologist Rajni Govindjee for her pioneering research in photosynthesis and retinal proteins on the occasion of her 80th birthday.