Diversification and spectral tuning in marine proteorhodopsins

Proteorhodopsins, ubiquitous retinylidene photoactive proton pumps, were recently discovered in the cosmopolitan uncultured SAR86 bacterial group in oceanic surface waters. Two related proteorhodopsin families were found that absorb light with different absorption maxima, 525 nm (green) and 490 nm (blue), and their distribution was shown to be stratified with depth. Using structural modeling comparisons and mutagenesis, we report here on a single amino acid residue at position 105 that functions as a spectral tuning switch and accounts for most of the spectral difference between the two pigment families. Furthermore, looking at natural environments, we found novel proteorhodopsin gene clusters spanning the range of 540-505 nm and containing changes in the same identified key switch residue leading to changes in their absorption maxima. The results suggest a simultaneous diversification of green proteorhodopsin and the new key switch variant pigments. Our observations demonstrate that this single-residue switch mechanism is the major determinant of proteorhodopsin wavelength regulation in natural marine environments.     

Mechanism of spectral tuning in green-absorbing proteorhodopsin

The absorption spectrum of green proteorhodopsin (GPR) is pH-dependent, exhibiting either red-shifted (low pH) or blue-shifted (high pH) absorption maxima. We examine the molecular basis of the pH-dependent spectral properties of green proteorhodopsin by using homology modeling and molecular orbital theory. Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII) are compared as homology templates. The model of GPR generated by using BR as the homology parent is better than that generated by using SRII on the basis of the potential energy, relative stability to dynamics, and ability to rationalize pH effects. MNDO-PSDCI (molecular neglect of differential overlap with partial single- and double-configuration interaction) calculations provide insight into the spectroscopic properties of GPR and help rule out the viability of the SRII-based model. The proximity of His 75 to the quadrupole residues (LYR, D97, D227, and R94) in the BR-based model provides a good model for both the low- and high-pH spectral states of GPR. The observation that BR is a better structural model for GPR than SRII is in contrast to our previous study of BPR, which observed that SRII was the better homology parent [Hillebrecht, J. R. (2006) Biochemistry 45, 1579-1590]. The implications of this observation are discussed.     

Cone monochromacy and visual pigment spectral tuning in wobbegong sharks

Much is known regarding the evolution of colour vision in nearly every vertebrate class, with the notable exception of the elasmobranchs. While multiple spectrally distinct cone types are found in some rays, sharks appear to possess only a single class of cone and, therefore, may be colour blind. In this study, the visual opsin genes of two wobbegong species, Orectolobus maculatus and Orectolobus ornatus, were isolated to verify the molecular basis of their monochromacy. In both species, only two opsin genes are present, RH1 (rod) and LWS (cone), which provide further evidence to support the concept that sharks possess only a single cone type. Examination of the coding sequences revealed substitutions that account for interspecific variation in the photopigment absorbance spectra, which may reflect the difference in visual ecology between these species.     

Evolution and spectral tuning of visual pigments in birds and mammals

Variation in the types and spectral characteristics of visual pigments is a common mechanism for the adaptation of the vertebrate visual system to prevailing light conditions. The extent of this diversity in mammals and birds is discussed in detail in this review, alongside an in-depth consideration of the molecular changes involved. In mammals, a nocturnal stage in early evolution is thought to underlie the reduction in the number of classes of cone visual pigment genes from four to only two, with the secondary loss of one of these genes in many monochromatic nocturnal and marine species. The trichromacy seen in many primates arises from either a polymorphism or duplication of one of these genes. In contrast, birds have retained the four ancestral cone visual pigment genes, with a generally conserved expression in either single or double cone classes. The loss of sensitivity to ultraviolet (UV) irradiation is a feature of both mammalian and avian visual evolution, with UV sensitivity retained among mammals by only a subset of rodents and marsupials. Where it is found in birds, it is not ancestral but newly acquired.     

Mechanistic insight into the photosensory versatility of DXCF cyanobacteriochromes

Cyanobacteriochromes (CBCRs) are photosensory proteins related to the red/far-red phytochromes. Like phytochromes, CBCRs use linear tetrapyrrole (bilin) chromophores covalently attached via a thioether linkage to a conserved Cys residue also found in plant and cyanobacterial phytochromes. Unlike almost all phytochromes, CBCRs require only an isolated GAF domain to undergo efficient, reversible photocycles that are responsible for their broad light sensing range, spanning the visible to the near ultraviolet (UV). Sensing of blue, violet, and near-UV light by CBCRs requires another Cys residue proposed to form a second linkage to the bilin precursor. Light triggers 15,16-double bond isomerization as in phytochromes. After photoisomerization, elimination of the second linkage frequently occurs, thus yielding a large red shift of the stable photoproducts. Here we examine this process for representative DXCF CBCRs, a large subfamily named for the conserved Asp-Xaa-Cys-Phe motif that contains their second Cys residue. DXCF CBCRs with such dual-Cys photocycles yield a wide diversity of photoproducts absorbing teal, green, or orange light. Using a combination of CD spectroscopy, chemical modification, and bilin substitution experiments with recombinant CBCRs from Thermosynechococcus elongatus and Nostoc punctiforme expressed in Escherichia coli, we establish that second-linkage elimination is required for all of these photocycles. We also identify deconjugation of the D-ring as the mechanism for specific detection of teal light, at approximately 500 nm. Our studies thus provide new mechanistic insight into the photosensory versatility of this important family of photosensory proteins.     

Molecular mechanism of spectral tuning in sensory rhodopsin II.

Sensory rhodopsin II (SRII) is unique among the archaeal rhodopsins in having an absorption maximum near 500 nm, blue shifted roughly 70 nm from the other pigments. In addition, SRII displays vibronic structure in the lambda(max) absorption band, whereas the other pigments display fully broadened band maxima. The molecular origins responsible for both photophysical properties are examined here with reference to the 2.4 A crystal structure of sensory rhodopsin II (NpSRII) from Natronobacterium pharaonis. We use semiempirical molecular orbital theory (MOZYME) to optimize the chromophore within the chromophore binding site, and MNDO-PSDCI molecular orbital theory to calculate the spectroscopic properties. The entire first shell of the chromophore binding site is included in the MNDO-PSDCI SCF calculation, and full single and double configuration interaction is included for the chromophore pi-system. Through a comparison of corresponding calculations on the 1.55 A crystal structure of bacteriorhodopsin (bR), we identify the principal molecular mechanisms, and residues, responsible for the spectral blue shift in NpSRII. We conclude that the major source of the blue shift is associated with the significantly different positions of Arg-72 (Arg-82 in bR) in the two proteins. In NpSRII, this side chain has moved away from the chromophore Schiff base nitrogen and closer to the beta-ionylidene ring. This shift in position transfers this positively charged residue from a region of chromophore destabilization in bR to a region of chromophore stabilization in NpSRII, and is responsible for roughly half of the blue shift. Other important contributors include Asp-201, Thr-204, Tyr-174, Trp-76, and W402, the water molecule hydrogen bonded to the Schiff base proton. The W402 contribution, however, is a secondary effect that can be traced to the transposition of Arg-72. Indeed, secondary interactions among the residues contribute significantly to the properties of the binding site. We attribute the increased vibronic structure in NpSRII to the loss of Arg-72 dynamic inhomogeneity, and an increase in the intensity of the second excited (1)A(g)(-) -like state, which now appears as a separate feature within the lambda(max) band profile. The strongly allowed (1)B(u)(+)-like state and the higher-energy (1)A(g)(-) -like state are highly mixed in NpSRII, and the latter state borrows intensity from the former to achieve an observable oscillator strength.     

Modeling spectral tuning in monomeric teal fluorescent protein mTFP1

We present results of theoretical studies of the variants of the monomeric teal fluorescent protein from Clavularia coral (mTFP1) which present promising members from the GFP family. Predictions of quantum chemical approaches including density functional theory and semiempirical approximations are presented for the model systems which mimic the chromophores in different environments. We describe the excitation energy spectrum of the cyan mTFP1 fluorescent protein with the original chromophore and with chromophore mutants Tyr67His and Tyr67Trp.     

Evolution and mechanism of spectral tuning of blue-absorbing visual pigments in butterflies

The eyes of flower-visiting butterflies are often spectrally highly complex with multiple opsin genes generated by gene duplication, providing an interesting system for a comparative study of color vision. The Small White butterfly, Pieris rapae, has duplicated blue opsins, PrB and PrV, which are expressed in the blue (λ _max = 453 nm) and violet receptors (λ _max = 425 nm), respectively. To reveal accurate absorption profiles and the molecular basis of the spectral tuning of these visual pigments, we successfully modified our honeybee opsin expression system based on HEK293s cells, and expressed PrB and PrV, the first lepidopteran opsins ever expressed in cultured cells. We reconstituted the expressed visual pigments in vitro, and analysed them spectroscopically. Both reconstituted visual pigments had two photointerconvertible states, rhodopsin and metarhodopsin, with absorption peak wavelengths 450 nm and 485 nm for PrB and 420 nm and 482 nm for PrV. We furthermore introduced site-directed mutations to the opsins and found that two amino acid substitutions, at positions 116 and 177, were crucial for the spectral tuning. This tuning mechanism appears to be specific for invertebrates and is partially shared by other pierid and lycaenid butterfly species.     

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 pigment's 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.     

The polymorphic photopigments of the marmoset: spectral tuning and genetic basis.

The marmoset (Callithrix jacchus jacchus), a South American monkey, is polymorphic for the middle- to long-wave cone photopigments: the three variant pigments have spectral peaks at 543, 556 and 563 nm. Comparisons of the deduced amino acid sequences of these pigments indicate that the variations in spectral sensitivity are associated with the presence or absence of hydroxyl-bearing residues at sites 180 and 285; but, in contrast to the additive hypothesis of Neitz et al. (1991), we propose that adjustments at site 233 may also be required to produce viable long-wave and middle-wave pigments. Within a family group of monkeys, we find that a restriction site polymorphism in the photopigment gene segregates in a way that is consistent with the single X-linked gene hypothesis previously proposed on the basis of the photopigment types present in male and female marmosets.     

Mechanisms of spectral tuning in the RH2 pigments of Tokay gecko and American chameleon

At present, molecular bases of spectral tuning in rhodopsin-like (RH2) pigments are not well understood. Here, we have constructed the RH2 pigments of nocturnal Tokay gecko (Gekko gekko) and diurnal American chameleon (Anolis carolinensis) as well as chimeras between them. The RH2 pigments of the gecko and chameleon reconstituted with 11-cis-retinal had the wavelengths of maximal absorption (lambda(max)'s) of 467 and 496 nm, respectively. Chimeric pigment analyses indicated that 76-86%, 14-24%, and 10% of the spectral difference between them could be explained by amino acid differences in transmembrane (TM) helices I-IV, V-VII, and amino acid interactions between the two segments, respectively. Evolutionary and mutagenesis analyses revealed that the lambda(max)'s of the gecko and chameleon pigments diverged from each other not only by S49A (serine to alanine replacement at residue 49), S49F (serine to phenylalanine), L52M (leucine to methionine), D83N (aspartic acid to asparagine), M86T (methionine to threonine), and T97A (threonine to alanine) but also by other amino acid replacements that cause minor lambda(max)-shifts individually.     

Spectrotemporal processing in spectral tuning modules of cat primary auditory cortex

Spectral integration properties show topographical order in cat primary auditory cortex (AI). Along the iso-frequency domain, regions with predominantly narrowly tuned (NT) neurons are segregated from regions with more broadly tuned (BT) neurons, forming distinct processing modules. Despite their prominent spatial segregation, spectrotemporal processing has not been compared for these regions. We identified these NT and BT regions with broad-band ripple stimuli and characterized processing differences between them using both spectrotemporal receptive fields (STRFs) and nonlinear stimulus/firing rate transformations. The durations of STRF excitatory and inhibitory subfields were shorter and the best temporal modulation frequencies were higher for BT neurons than for NT neurons. For NT neurons, the bandwidth of excitatory and inhibitory subfields was matched, whereas for BT neurons it was not. Phase locking and feature selectivity were higher for NT neurons. Properties of the nonlinearities showed only slight differences across the bandwidth modules. These results indicate fundamental differences in spectrotemporal preferences--and thus distinct physiological functions--for neurons in BT and NT spectral integration modules. However, some global processing aspects, such as spectrotemporal interactions and nonlinear input/output behavior, appear to be similar for both neuronal subgroups. The findings suggest that spectral integration modules in AI differ in what specific stimulus aspects are processed, but they are similar in the manner in which stimulus information is processed.     

Computational strategy for tuning spectral properties of red fluorescent proteins

Computational methods of quantum chemistry are used to characterize structures and vertical excitation energies of the S(0)-S(1) optical transitions in the chromophore binding pockets of the red fluorescent proteins DsRed and of its artificial mutant mCherry. As previously shown, optimizing the equilibrium geometry configurations with B3LYP density functional theory, followed by ZINDO calculations of the electronic excitations, yields positions of the optical bands in good agreement with experimental data. These large scale quantum calculations elucidate the role of the hydrogen bonded network as well as point mutations in the absorption spectra of the DsRed and mCherry proteins. The effect of an external electric field applied to the fluorescent protein chromophores is examined and shows that such fields may result in large shifts in spectral bands. These strategies can be applied for rational design of the fluorescent proteins by site-directed mutagenesis.     

Thiol-based photocycle of the blue and teal light-sensing cyanobacteriochrome Tlr1999

Cyanobacteriochromes are a spectrally diverse photoreceptor family that binds a bilin chromophore. For some cyanobacteriochromes, in addition to the widely conserved cysteine to anchor the chromophore, its ligation with a second cysteine is responsible for a remarkable blue shift. Herein, we report a newly discovered cyanobacteriochrome Tlr1999 exhibiting reversible photoconversion between a blue-absorbing form at 418 nm (P418) and a teal-absorbing form at 498 nm (P498). Acidic denaturation suggests that P418 harbors C15-Z phycoviolobilin, whereas P498 harbors C15-E phycoviolobilin. When treated with iodoacetamide, which irreversibly modifies thiol groups, P418 is slowly converted to a green-absorbing photoinactive form denoted P552. The absorption spectrum of P498 appears to be unaffected by iodoacetamide, but when iodoacetamide modified, it is photoconverted to P552. These results suggest that a covalent bond exists between the second Cys and the phycoviolobilin in P418 but not in P498. Subsequent treatment with dithiothreitol converts P552 into P418, whereas dithiothreitol reduces P498 to yield P420, a photoinactive form. Site-directed mutagenesis shows that the second Cys is essential for assembly of the photoactive holoprotein and that the photoactivity of this inert mutant is partially rescued by β-mercaptoethanol. These results suggest that the covalent attachment and detachment of a thiol, although not necessarily that of the second Cys, is critical for the reversible spectral blue shift and the complete photocycle. We propose a thiol-based photocycle, in which the thiol-modified P552 and P420 are intermediate-like forms.     

Rod opsin sequence in the John Dory: further evidence for the spectral tuning of rhodopsin

The opsin from the John Dory Zeus faber rod visual pigment (maximum spectral sensitivity =492 nm) was cloned and sequenced. Comparison of the John Dory rod opsin sequence with those from other fish with blue-shifted scotopic spectral sensitivity provides further evidence for the spectral tuning of this group of visual pigments.     

The role of charge-transfer states in the spectral tuning of antenna complexes of purple bacteria

Local damage (mainly burning, heating, and mechanical wounding) induces propagation of electrical signals, namely, variation potentials, which are important signals during the life of plants that regulate different physiological processes, including photosynthesis. It is known that the variation potential decreases the rate of CO₂ assimilation by the Calvin–Benson cycle; however, its influence on light reactions has been poorly investigated. The aim of our work was to investigate the influence of the variation potential on the light energy flow that is absorbed, trapped and dissipated per active reaction centre in photosystem II and on the flow of electrons through the chloroplast electron transport chain. We analysed chlorophyll fluorescence in pea leaves using JIP-test and PAM-fluorometry; we also investigated delayed fluorescence. The electrical signals were registered using extracellular electrodes. We showed that the burning-induced variation potential stimulated a nonphotochemical loss of energy in photosystem II under dark conditions. It was also shown that the variation potential gradually increased the flow of light energy absorbed, trapped and dissipated by photosystem II. These changes were likely caused by an increase in the fraction of absorbed light distributed to photosystem II. In addition, the variation potential induced a transient increase in electron flow through the photosynthetic electron transport chain. Some probable mechanisms for the influence of the variation potential on the light reactions of photosynthesis (including the potential role of intracellular pH decrease) are discussed in the work.     

Molecular basis of spectral tuning in the red- and green-sensitive (M/LWS) pigments in vertebrates

Vertebrate vision is mediated by five groups of visual pigments, each absorbing a specific wavelength of light between ultraviolet and red. Despite extensive mutagenesis analyses, the mechanisms by which contemporary pigments absorb variable wavelengths of light are poorly understood. We show that the molecular basis of the spectral tuning of contemporary visual pigments can be illuminated only by mutagenesis analyses using ancestral pigments. Following this new principle, we derive the "five-sites" rule that explains the absorption spectra of red and green (M/LWS) pigments that range from 510 to 560 nm. Our findings demonstrate that the evolutionary method should be used in elucidating the mechanisms of spectral tuning of four other pigment groups and, for that matter, functional differentiations of any other proteins.     

Fine tuning of the spectral properties of LH2 by single amino acid residues

The peripheral light-harvesting complex, LH2, of Rhodobacter sphaeroides consists of an assembly of membrane-spanning alpha and beta polypeptides which assemble the photoactive bacteriochlorophyll and carotenoid molecules. In this study we systematically investigated bacteriochlorophyll-protein interactions and their effect on functional bacteriochlorophyll assembly by site-directed mutations of the LH2 alpha-subunit. The amino acid residues, isoleucine at position -1 and serine at position -4 were replaced by 12 and 13 other residues, respectively. All residues replacing isoleucine at position -1 supported the functional assembly of LH2. The replacement of isoleucine by glycine, glutamine or asparagine, however, produced LH2 complex with significantly altered spectral properties in comparison to LH2 WT. As indicated by resonance Raman spectroscopy extensive rearrangement of the bacteriochlorophyll-B850 macrocycle(s) took place in LH2 in which isoleucine -1 was replaced by glycine. The replacement results in disruption of the H-bond between the C3 acetyl groups and the aromatic residues +13/+14 without affecting the H-bond involving the C13(1) keto group. In contrast, nearly all amino acid replacements of serine at position -4 resulted in shifting of the bacteriochlorophyll-B850 red most absorption maximum. Interestingly, the extent of shifting closely correlated with the volume of the residue at position -4. These results illustrate that fine tuning of the spectral properties of the bacteriochlorophyll-B850 molecules depend on their packing with single amino acid residues at distinct positions.     

Wide-band spectral tuning of heat receptors in the pit organ of the copperhead snake (Crotalinae)

Receptors located in the facial pit organ of certain species of snake signal the presence of prey. Infrared radiation is an effective stimulus suggesting that these receptors may be low-threshold temperature receptors. We recorded from the nerve innervating the pit organ of snakes belonging to the family of Crotalinae while stimulating the receptive area with well-defined optical stimuli. The objective was to determine the sensitivity of these receptors to a wide range (0.400-10.6 micro m) of optical stimuli to determine if a temperature-sensitive or photosensitive protein initiated signal transduction. We found that receptors in the pit organ exhibited a unique broad response to a wide range of electromagnetic radiation ranging from the near UV to the infrared. The spectral tuning of these receptors parallels closely the absorption spectra of water and oxyhemoglobin, the predominant chromophore in tissue. Our results support the hypothesis that these are receptors activated by minute temperature changes induced by direct absorption of optical radiation in the thin pit organ membrane.