The effect of anions on spectral properties of iodopsin and native cones in the frog retina (microspectrophotometric study)

Absorption spectra of single outer segments of the frog Rana temporaria photoreceptors were registered. Effects of nitrate and chloride ions on spectral properties of cone and rod pigments were compared. These pigments proved to differ in structure of the native photoreceptor membrane and, therefore, in effect of hydrophile environment on the chromophore centrum. Substitution of chloride by nitrate ions led to the hypochromic shift of the cone absorption spectrum (20-25 nm) but does not affect the spectrum on case of rod pigment. The ionochromic behaviour of cone pigments resembles that of the light-sensitive halobacterium protein halorhodopsin, in native membrane. We suppose that the effect of anions on the chromophore centrum may be the cause of bathochromic shifts of absorption spectra of longwave-length retinal-containing pigments.     

A dark and constitutively active mutant of the tiger salamander UV pigment

A triple mutant (F86L/T93P/S118T; bovine rhodopsin numbering) of the tiger salamander UV cone pigment appears to be trapped in an open conformation that is metarhodopsin-II-like. The pigment is able to activate transducin in the dark, and the ligand-free apoprotein is also able to activate transducin constitutively. The pigment permits protons and chloride ions from solution access to the active site as it displays a pH- and NaCl-dependent absorption spectrum not observed with the wild-type pigment. However, the wild-type properties of light-dependent activity and a pH-independent absorption spectrum are recovered upon reconstitution of the triple mutant with 11-cis-9-demethyl retinal. These results suggest that binding the native chromophore cannot deactivate the protein because of steric interactions between the protein, possibly residue 118, and the 9-methyl group of the chromophore. Furthermore, the absorption spectrum of the 9-demethyl retinal regenerated pigment exhibits a band broader and with lower extinction at the absorption maximum than either the human blue or salamander UV wild-type pigments generated with the same retinal analogue. The broad spectrum appears to be comprised of two or more species and can be well-fit by a sum of scaled spectra of the two wild-type pigments. Binding the chromophore appears to trap the pigment in two or more conformations. The triple mutant reported here represents the first example of a dark-active cone pigment and constitutively active cone opsin.     

Circular dichroism spectroscopy of fluorescent proteins

Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red fluorescent protein from the coral species Discosoma (DsRed). We demonstrate that CD spectra in the spectral fingerprint region of the chromophore yield spectra that after normalization are not coincident with the normalized absorbance spectra of GFP, YFP and DsRed. On the other hand, the CD spectra of BFP and CFP coincide with the absorbance spectra. The resolution of absorption and CD spectra into Gaussian bands confirmed the location of the different electronic band positions of GFP and YFP as reported in the literature using other techniques. In the case of BFP and CFP the location of Gaussian bands provided information of the vibrational progression of the electronic absorption bands. The CD spectrum of DsRed is anomalous in the sense that the major CD band has a clear excitonic character. Far-UV CD spectra of GFP confirmed the presence of the high beta-sheet content of the polypeptide chain in the three-dimensional structure.     

Isolation and properties of a fluorescent compound, factor 420 , from Methanobacterium strain M.o.H

A new fluorescent compound, factor(420) (F(420)), which is involved in the hydrogen metabolism of hydrogen-grown Methanobacterium strain M.o.H. has been isolated and purified. Acid hydrolysis of this compound with 6 m HCl for 24 hr releases a ninhydrin-positive compound (glutamic acid), an acid-stable chromophore, phosphate, and an ether-soluble phenolic component. Factor(420) may be reduced by either sodium dithionite or sodium borohydride at pH 7.3 with concomitant loss of its fluorescence and its major absorption peak at 420 nm. Crude cell-free extracts of strain M.o.H. reduce F(420) only under a hydrogen atmosphere. F(420) is photolabile aerobically in neutral and basic solutions, whereas the acid-stable chromophore is not photolabile under these conditions. An approximate molecular weight of 630 +/- 8% for F(420) was determined by Sephadex G-25 chromatography. At the present time, F(420) is proposed as a trivial name for the unknown fluorescent compound because of its strong absorption maximum of 420 nm at pH 7.     

Deletion mapping of the Aequorea victoria green fluorescent protein

Aequorea victoria green fluorescent protein (GFP) is a promising fluorescent marker which is active in a diverse array of prokaryotic and eukaryotic organisms. A key feature underlying the versatility of GFP is its capacity to undergo heterocyclic chromophore formation by cyclization of a tripeptide present in its primary sequence and thereby acquiring fluorescent activity in a variety of intracellular environments. In order to define further the primary structure requirements for chromophore formation and fluorescence in GFP, a series of N- and C-terminal GFP deletion variant expression vectors were created using the polymerase chain reaction. Scanning spectrofluorometric analyses of crude soluble protein extracts derived from eleven GFP expression constructs revealed that amino acid (aa) residues 2–232, of a total of 238 aa in the native protein, were required for the characteristic emission and absorption spectra of native GFP. Heterocyclic chromophore formation was assayed by comparing the absorption spectrum of GFP deletion variants over the 300–500-nm range to the absorption spectra of full-length GFP and GFP deletion variants missing the chromophore substrate domain from the primary sequence. GFP deletion variants lacking fluorescent activity showed no evidence of heterocyclic ring structure formation when the soluble extracts of their bacterial expression hosts were studied at pH 7.9. These observations suggest that the primary structure requirements for the fluorescent activity of GFP are relatively extensive and are compatible with the view that much of the primary structure serves an autocatalytic function.     

How color visual pigments are tuned.

The absorption maximum of the retinal chromophore in color visual pigments is tuned by interactions with the protein (opsin) to which it is bound. Recent advances in the expression of rhodopsin-like transmembrane receptors and in spectroscopic techniques have allowed us to measure resonance Raman vibrational spectra of the retinal chromophore in recombinant visual pigments to examine the molecular basis of this spectral tuning. The dominant physical mechanism responsible for the opsin shift in color vision is the interaction of dipolar amino acid residues with the ground- and excited-state charge distributions of the chromophore.     

Electronic spectroscopy and solvatochromism in the chromophore of GFP and the Y66F mutant.

The electronic spectra of the chromophore of the wild type green fluorescent protein, GFP, and of a mutant form Y66F GFP in which the chromophore lacks the hydroxyl group have been studied. The acid-base properties, solvatochromism, vibronic structure and edge excitation red shift have all been measured. The results are compared with the spectra of the chromophore in the protein environment. These data suggest that the transition energy for the GFP chromophore is influenced by a number of factors in its environment, and in particular by hydrogen bonding.     

A purple-blue chromoprotein from Goniopora tenuidens belongs to the DsRed subfamily of GFP-like proteins

A number of recently cloned chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well. Here we report that a far-red chromoprotein from Goniopora tenuidens (gtCP) contains a chromophore of the same chemical structure as DsRed. Denaturation kinetics of both DsRed and gtCP under acidic conditions indicates that the red form of the chromophore (absorption maximum at 436 nm) converts to the GFP-like form (384 nm) by a one-stage reaction. Upon neutralization, the 436-nm form of gtCP, but not the 384-nm form, renaturates instantly, implying that the former includes a chromophore in its intact state. gtCP represents a single-chain protein and, upon harsh denaturing conditions, shows three major bands in SDS/PAGE, two of which apparently result from hydrolysis of an acylimine C=N bond. Instead of having absorption maxima at 384 nm and 450 nm, which are characteristic for a GFP-like chromophore, fragmented gtCP shows a different spectrum, which presumably corresponds to a 2-keto derivative of imidazolidinone. Mass spectra of the chromophore-containing peptide from gtCP reveal an additional loss of 2 Da relative to the GFP-like chromophore. Tandem mass spectrometry of the chromopeptide shows that an additional bond is dehydrogenated in gtCP at the same position as in DsRed. Altogether, these data suggest that gtCP belongs to the same subfamily as DsRed (in the classification of GFP-like proteins based on the chromophore structure type).     

Probing the ground state structure of the green fluorescent protein chromophore using Raman spectroscopy

We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65T mutant of this intrinsically fluorescent protein together with data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate . In the pH range 1-14, the model compound has two macroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ring nitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our results do not provide any evidence for the zwitterionic form of the chromophore, with the phenolic group deprotonated and the imidazolinone ring nitrogen protonated, being present in the GFP proteins. In addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It is found that the ground state structure of the anionic form of the chromophore, which is most relevant to the fluorescent properties, is strongly dependent on the chromophore environment whereas the neutral form seems to be insensitive. A linear correlation between the absorption properties and the ground state structure is demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm(-1).     

Spectroscopic characterization of the photocycle intermediates of photoactive yellow protein.

The absorption spectra of photocycle intermediates of photoactive yellow protein mutants were compared with those of the corresponding intermediates of wild type to probe which amino acid residues interact with the chromophore in the intermediate states. B and H intermediates were produced by irradiation and trapped at 80 K, and L intermediates at 193 K. The absorption spectra of these intermediates produced from R52Q were identical to those from wild type, whereas those from E46Q and T50V were 7-15 nm red-shifted as those in the dark states. The absorption spectra of M intermediates were measured by flash photolysis at room temperature. Those of Y42F, T50V, and R52Q were identical to that of wild type, whereas that of E46Q was 11 nm red-shifted. Assuming that the intermediates of mutants have a structure comparable to that of wild type, these findings suggest the following: Glu46 interacts with the chromophore throughout the photocycle, interaction between the chromophore and Thr50 as well as Tyr42 is lost upon the formation of M intermediate, and Arg52 never interacts with the chromophore directly. The hydrogen-bonding network around the phenolic oxygen of the chromophore would be thus maintained until L intermediate decays, and the global conformational change would take place by the loss of the hydrogen bond between the chromophore and Tyr42. This model conflicts with some of the results of previous crystallographic studies, suggesting that the reaction mechanism in the crystal may be different from that in solution.     

Bathoproducts of rhodopsin, isorhodopsin I, and isorhodopsin II.

Bathorhodopsins were prepared by partially (10--15%) photoconverting bovine rhodopsin (11-cis chromophore) or isorhodopsin I (9-cis chromophore) at 77 degrees K; care was taken to avoid establishing photostationary states. The absorption spectra calculated for the bathorhodopsins derived from the two parent pigments are identical in their lambda max 'S, bandwidths, and extinction coefficients. This result provides further support for the hypothesis that bathorhodopsin is a common intermediate between an 11-cis pigment (rhodopsin) and a 9-cis one (isorhodopsin I) and thus probably has an all-trans chromophore. This in turn is strong evidence for the cis-trans isomerization model of the primary event in vision. The spectrum of the bathoproduct of isorhodopsin II (9,13-dicis chromophore) is different from the other pigments' bathoproducts.     

Multiple visual pigments in a photoreceptor of the salamander retina

Although a given retina typically contains several visual pigments, each formed from a retinal chromophore bound to a specific opsin protein, single photoreceptor cells have been thought to express only one type of opsin. This design maximizes a cell''s sensitivity to a particular wavelength band and facilitates wavelength discrimination in retinas that process color. We report electrophysiological evidence that the ultraviolet-sensitive cone of salamander violates this rule. This cell contains three different functional opsins. The three opsins could combine with the two different chromophores present in salamander retina to form six visual pigments. Whereas rods and other cones of salamander use both chromophores, they appear to express only one type of opsin per cell. In visual pigment absorption spectra, the bandwidth at half-maximal sensitivity increases as the pigment''s wavelength maximum decreases. However, the bandwidth of the UV-absorbing pigment deviates from this trend; it is narrow like that of a red-absorbing pigment. In addition, the UV-absorbing pigment has a high apparent photosensitivity when compared with that of red- and blue-absorbing pigments and rhodopsin. These properties suggest that the mechanisms responsible for spectrally tuning visual pigments separate two absorption bands as the wavelength of maximal sensitivity shifts from UV to long wavelengths.     

Spectral tuning of deep red cone pigments

Visual pigments are G-protein-coupled receptors that provide a critical interface between organisms and their external environment. Natural selection has generated vertebrate pigments that absorb light from the far-UV (360 nm) to the deep red (630 nm) while using a single chromophore, in either the A1 (11- cis-retinal) or A2 (11- cis-3,4-dehydroretinal) form. The fact that a single chromophore can be manipulated to have an absorption maximum across such an extended spectral region is remarkable. The mechanisms of wavelength regulation remain to be fully revealed, and one of the least well-understood mechanisms is that associated with the deep red pigments. We investigate theoretically the hypothesis that deep red cone pigments select a 6- s- trans conformation of the retinal chromophore ring geometry. This conformation is in contrast to the 6- s- cis ring geometry observed in rhodopsin and, through model chromophore studies, the vast majority of visual pigments. Nomographic spectral analysis of 294 A1 and A2 cone pigment literature absorption maxima indicates that the selection of a 6- s- trans geometry red shifts M/LWS A1 pigments by approximately 1500 cm (-1) ( approximately 50 nm) and A2 pigments by approximately 2700 cm (-1) ( approximately 100 nm). The homology models of seven cone pigments indicate that the deep red cone pigments select 6- s- trans chromophore conformations primarily via electrostatic steering. Our results reveal that the generation of a 6- s- trans conformation not only achieves a significant red shift but also provides enhanced stability of the chromophore within the deep red cone pigment binding sites.     

Mechanism of fluorescent cocoon sex identification for silkworms Bombyx mori

By using silkworms, Bombyx mori, fluorescent cocoon sex identification (FCSI) as an experimental material, direct fluorescence spectrometry of the cocoon surface indicates that the fluorescent color of silkworm cocoons is made up of two peaks of yellow and blue-purple fluorescence emission. The fluorescent difference between male and female cocoons is attributed to the differential absorption of yellow fluorescent substances by the midgut tissue of 5th instar female silkworms. Thin layer chromatography (TLC) and fluorescent spectra indicate that blue-purple fluorescent substances are composed of at least five blue-purple fluorescent pigments, and yellow fluorescent substances are made up of at least three. UV spectra and AlCl₃ color reaction show that the three fluorescent yellow pigments are flavonoids or their glycosides. Silkworm FCSI is due to selective absorption or accumulation of the yellow fluorescent pigments by the posterior midgut cells of female 5th instar larvae. The cells of the FCSI silkworm midgut, especially the cylinder intestinal cells of the posterior midgut have a component which is a yellow fluorescent pigment-specific binding protein that may be vigorously expressed in the 5th instar larvae.     

Resonance Raman characterization of Rhodobacter sphaeroides reaction centers bearing site-directed mutations at tyrosine M210

Resonance Raman (RR) spectroscopy and low-temperature absorption spectroscopy have been used to investigate the structural changes in the reaction centers (RCs) of Rhodobacter sphaeroides induced by site-specific mutations on the tyrosine (Y) M210 residue. RCs in which Y M210 has been genetically replaced with phenylalanine (F) or leucine (L) exhibit a 5-fold decrease in their primary electron-transfer kinetics (Finkele et al., 1990). The general similarity of RR spectra of the wild-type RCs as compared to those of the two mutant RCs indicates that no significant global structural changes occur upon mutation at the level of any of the six bacteriochlorin pigments. In the RR spectra of the two mutant RCs there is a conspicuous absence of contributions from the BPheM prosthetic group, which is interpreted in terms of a change in the resonance enhancement conditions of this chromophore. Low-temperature adsorption spectroscopy reveals marked shifts in the Qx absorption band of BPheM. This shift is interpreted as arising from a destabilization of the protein in the vicinity of BPheM and accounts for the change in resonance condition for this chromophore in its RR contributions. As well, there is a 3-nm red shift of the Qy absorption band of the BChls from 803 to 806 nm for the mutant RCs. Difference RR spectra yielding structural information concerning, selectively, the primary donor (P) indicate that the structure of the P binding pocket is conserved for these mutant RCs. The tyrosine M210 is not observed to be engaged in a hydrogen bond with either of the acetyl or keto carbonyls of P.     

Polarized absorption spectra of green fluorescent protein single crystals: transition dipole moment directions

Polarized absorption spectra of orthorhombic crystals of wild-type green fluorescent protein (GFP) were measured between 350 and 520 nm to obtain information on the directions of the electronic transition dipole moments ((-->)m) of the chromophore relative to the molecular axes. The transition dipole moment orientation is a basic spectroscopic parameter of relevance to biologists when interpreting F?rster-type fluorescence resonance energy transfer data and for comparing absorbance and fluorescence spectra of GFP with quantum chemical calculations. Maximal extinction was obtained throughout the spectrum when the polarization direction of the electric vector of incident light was parallel to the c-axis of the crystal. The transition dipole moments were assumed to be parallel to the plane of the chromophore. With this assumption and the measured dichroic ratios in the crystals, the transition dipole moments associated with the neutral (lambda(max) = 398 nm) and anionic (lambda(max) = 478 nm) forms of the chromophore were found to subtend angles of approximately 26 degrees and 13 degrees (counterclockwise), respectively, with the vector that joins the phenolic and imidazolinone oxygen atoms of the chromophore.     

Backbone dynamics of green fluorescent protein and the effect of histidine 148 substitution

Green fluorescent protein (GFP) and its mutants have become valuable tools in molecular biology. GFP has been regarded as a very stable and rigid protein with the beta-barrel shielding the chromophore from the solvent. Here, we report the 15N nuclear magnetic resonance (NMR) studies on the green fluorescent protein (GFPuv) and its mutant His148Gly. 15N NMR relaxation studies of GFPuv show that most of the beta-barrel of GFP is rigid on the picosecond to nanosecond time scale. For several regions, including the first alpha-helix and beta-sheets 3, 7, 8, and 10, increased hydrogen-deuterium exchange rates suggest a substantial conformational flexibility on the microsecond to millisecond time scales. Mutation of residue 148 located in beta-sheet 7 is known to have a strong impact on the fluorescence properties of GFPs. UV absorption and fluorescence spectra in combination with 1H-15N NMR spectra indicate that the His148Gly mutation not only reduces the absorption of the anionic chromophore state but also affects the conformational stability, leading to the appearance of doubled backbone amide resonances for a number of residues. This suggests the presence of two conformations in slow exchange on the NMR time scale in this mutant.     

Spectrophotometric measurement of carotenes, xanthophylls, and chlorophylls in extracts from plant seeds

The method of spectrophotometric measurement of carotenoid and chlorophyll content in extracts from plant seeds was modified. The pigments were extracted with a mixture of petroleum ether (PE) and tetrahydrofuran (THF) (PE to THF ratio 4: 1). Equations for calculations of β-carotene, lutein, chlorophyll a and b content in PE: THF mixture were obtained using specific absorption coefficients of solutions of particular pigments. It was shown that subtraction of chlorophyll contribution from the absorption spectrum of seed extracts yields spectra, which could be used in some cases for calculation of carotene and xanthophyll contents.     

Purification from baker's yeast of an activator of DNA photolyase.

The activity of purified DNA photolyase from Baker's yeast is enhanced by a compound (Activator (III)) obtained from yeast by chloroform extraction ion exchange chromatography and gel filtration. Thin layer chromatography and spectral data indicate that the compound is homogeneous. Activator III emits at 350 and 440 nm when excited at 290 nm, and emits at 440 nm when excited at 358 nm. After acid hydrolysis, emission at 440 nm is produced only by excitation at 358 nm, indicating that activator (III) contains two separate chromophoric moieties. The chromophore excited by 358 nm light has a pK of 9-11, while the other chromophore has a pK of 4-5, and possibly of 9-11. The enhancement of photolytic activity by activator (III) at a concentration equimolar with that of the enzyme and the similarity of the fluorescent spectra of the activator with that of heat-denatured photolyase, suggests that the activator may be the chromophore associated with the enzyme.     

Spectroscopic study of the batho-to-lumi transition during the photobleaching of rhodopsin using ring-modified retinal analogues

Photochemical and subsequent thermal reactions of rhodopsin containing 9-cis-retinal [Rh(9)] or one of four analogues with 9-cis geometries formed from ring-modified retinals, alpha-retinal [alpha Rh(9)], acyclic retinal [AcRh(9)], acyclic alpha-retinal [Ac alpha Rh(9)], and 5-isopropyl-alpha-retinal [P alpha Rh(9)] were investigated by low-temperature spectrophotometry and nanosecond laser photolysis. Irradiation of each pigment at -180 degrees C produced a photosteady-state mixture containing the original 9-cis pigment, its 11-cis pigment, and a photoproduct, indicating that the primary process of each pigment is a photoisomerization of its chromophore. The photoproduct produced by the irradiation of AcRh(9) had an absorption spectrum red shifted from the original AcRh(9) and was identified as the batho intermediate of AcRh(9). It was converted to the lumi intermediate through a metastable species, the BL intermediate, which has never been detected in Rh(9) at low temperature and whose absorption maximum was at shorter wavelengths than that of the batho intermediate. In contrast, the absorption maxima of the photoproducts produced from the other analogue pigments were at shorter wavelengths than those of the original pigments. They were identified as BL intermediates on the basis of their absorption maxima and thermal stabilities. The formation time constant of the lumi intermediate at room temperature was found to be dependent on the extent of modification of the ring portion of the chromophore, decreasing with the complete truncation of the cyclohexenyl ring [Ac alpha Rh(9)] and increasing with the attachment of the isopropyl group to the ring [P alpha Rh(9)].(ABSTRACT TRUNCATED AT 250 WORDS)