Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant

The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms. GFP emits bright green light (λ_max = 510 nm) when excited with ultraviolet (UV) or blue light (λ_max = 395 nm, minor peak at 470 nm). The chromophore in GFP is intrinsic to the primary structure of the protein, and fluorescence from GFP does not require additional gene products, substrates or other factors. GFP fluorescence is stable, species-independent and can be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry [Chalfie et al., Science 263 (1994) 802–805; Stearns, Curr. Biol. 5 (1995) 262–264]. The protein appears to undergo an autocatalytic reaction to create the fluorophore [Heim et al., Proc. Natl. Acad. Sci. USA 91 (1994) 12501–12504] in a process involving cyclization of a Tyr⁶⁶ aa residue. Recently [Delagrave et al., Bio/Technology 13 (1995) 151–154], a combinatorial mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A. victoria GFP, yielding a number of different mutants with redshifted fluorescence excitation spectra. One of these, RSGFP4, retains the characteristic green emission spectra (λ_max = 505 nm), but has a single excitation peak (λ_max = 490 nm). The fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP from the sea pansy, Renilla reniformis [Ward and Cormier, Photobiochem. Photobiol. 27 (1978) 389–396]. In the present study, we demonstrate by fluorescence microscopy that selective excitation of A. victoria GFP and RSGFP4 allows for spectral separation of each fluorescent signal, and provides the means to image these signals independently in a mixed population of bacteria or mammalian cells.     

Photolysis of the ion pair triphenylsulfonium thiophenolate induced by outersphere charge transfer excitation

The ion pair Ph₃S⁺PhS⁻ can be viewed as a sulfur-based mixed-valence system which is characterized by a PhS⁻ to Ph₃S⁺ outersphere charge transfer (OSCT) absorption at λ_max=390 nm (in CHCl₃). OSCT excitation leads to the radical pair Ph₃S/PhS which undergoes subsequent elimination and radical coupling processes with the final formation of Ph₂S. The photolysis proceeds with a quantum yield of φ=0.23 at λ_irr=436 nm.     

Chromcrphore-Environment Interaction of Visual Pigments in Model System

SEVERAL laboratories^1–6 have recently been concerned with the mechanism of the bathochromic shift of about 120 nm which results when 11-cis retinal (λ _max 380 nm) combines with the protein opsin to form rhodopsin (λ_max 498 nm). A red shift of up to 186 nm is involved in the formation of iodopsin from 11-cis retinal and cone opsin^7,8. The active site of bovine rhodopsin consists of the 11-cis retinylidene chromophore attached to a primary amine group of the protein forming a Schiff-base linkage of the type shown in Fig. 1, Ia. On the basis of the chemical reactions of rhodopsin and its derivatives it has been suggested that an interaction between a protonated form of the chromophore (structure of the type Ib) and a lipophilic environment contributes¹¹ to the red shift.     

Absorption spectrum of cattle hypsorhodopsin

On irradiation at liquid helium temperatures, rhodopsin is converted into hypsorhodopsin which decays to bathorhodopsin above 23 K. The absorption spectrum of cattle hypsorhodopsin (λ_max = 435 nm) is found to include a new sideband around 540 nm. This sideband may be due to π^* ← π transition to ¹A^?_g like state, which is made partially allowed by distortion of polyene chain of the retinylidene chromophore.     

Different Visible Colors and Green Fluorescence Were Obtained from the Mutated Purple Chromoprotein Isolated from Sea Anemone

Green fluorescent protein (GFP)-like proteins have been studied with the aim of developing fluorescent proteins. Since the property of color variation is understudied, we isolated a novel GFP-like chromoprotein from the carpet anemone Stichodactyla haddoni, termed shCP. Its maximum absorption wavelength peak (λ _max) is located at 574 nm, resulting in a purple color. The shCP protein consists of 227 amino acids (aa), sharing 96 % identity with the GFP-like chromoprotein of Heteractis crispa. We mutated aa residues to examine any alteration in color. When E63, the first aa of the chromophore, was replaced by serine (E63S), the λ _max of the mutated protein shCP-E63S was shifted to 560 nm and exhibited a pink color. When Q39, T194, and I196, which reside in the surrounding 5 Å of the chromophore’s microenvironment, were mutated, we found that (1) the λ _max of the mutated protein shCP-Q39S was shifted to 518 nm and exhibited a red color, (2) shCP-T194I exhibited a purple-blue color, and (3) an additional mutation at I196H of the mutated protein shCP-E63L exhibited green fluorescence. In contrast, when the aa located neither at the chromophore nor within its microenvironment were mutated, the resultant proteins shCP-L122H, -E138G, -S137D, -T95I, -D129N, -T194V, -E138Q, -G75E, -I183V, and -I70V never altered their purple color, suggesting that mutations at the shCP chromophore and the surrounding 5 Å microenvironment mostly control changes in color expression or cause fluorescence to develop. Additionally, we found that the cDNAs of shCP and its mutated varieties are faithfully and stably expressed both in Escherichia coli and zebrafish embryos.     

Internally illuminated photobioreactor using a novel type of light-emitting diode (LED) bar for cultivation of <Emphasis Type="Italic">Arthrospira platensis</Emphasis>

The biochemical properties of Spirulina platensis in an internally illuminated photobioreactor (IlPBR) were investigated under different light-emitted diode (LED) wavelengths; blue (λ_max= 450 and 460 nm), green (λ_max= 525 nm), red (λ_max = 630 and 660 nm), and white (6,500K), with various light intensities (200, 500, 1,000, and 2,000 μmol/m²/sec) were examined. The highest specific growth rate, maximum biomass, and phycocyanin productivity occurred under the red LEDs (0.39/day, 0.10 g/L/day, and 0.14 g/g-cell/day, respectively) at 1,000 μmol/m²/sec; the lowest growth rate was obtained under blue LEDs. Indeed, the size of trichomes was changed into short form under blue LEDs at all light intensities or all LEDs at 2,000 μmol/m²/sec for the first 2 days after inoculation, and S. platensis did not grow in the IlPBR under the dark condition. These results provide a base for different approaches for designing the pilot scale photobioreactor and developing cost-effective light sources.     

The synthesis of triethylphosphine gold(I) 4-nitrobenzenethiolate and solvent dependent visible absorption spectra of 4-nitrobenzenethiolate

The synthesis of triethylphosphine gold(I) 4-nitrobenzenethiolate, Et₃PAu(SC₆H₄NO₂-4), is reported. Et₃PAu(SC₆H₄NO₂-4) displays a low energy visible electronic absorption band which is solvent dependent: EtOH (λ_max = 385 nm), acetonitrile (λ_max = 391 nm), THF (λ_max = 395 nm), and DMSO (λ_max = 402 nm). The corresponding low energy visible electronic absorption band of 4-nitrobenzenethiolate, 4-NO₂C₆H₄S⁻ also shows solvent dependency: acetonitrile, (λ_max = 484 nm), DMSO (λ_max = 502 nm), dimethylformamide (λ_max = 505 nm). The positive solvatochromic shifts for Et₃PAu(SC₆H₄NO₂-4) and 4-NO₂C₆H₄S⁻ are consistent with an intraligand (IL) charge transfer transition, i.e. π(S) → ∗π (C₆H₄NO₂-4) or n(S) → ∗π (C₆H₄NO₂-4). Assignment of 4-NO₂C₆H₄S⁻ was aided by a DFT calculation.     

Phosphorescence of gadolinium(III) chelates under ambient conditions

The gadolinium(III) chelates Gd(dtpaH₂), Gd(hfac)₃, Gd(tta)₃ and Gd(qu)₃ with dtpa=1,1,4,7,7-diethylenetriaminepentaacetate, hfac=hexafluoroacetylacetonate, tta=thenoyltrifluoroacetonate and qu=8-quinolinolate (or oxinate) show a phosphorescence under ambient conditions. While the UV emission of Gd(dtpaH₂) at λ_max=312 nm comes from a metal-centered ff state, the bluish (λ_max=462 nm), green (λ_max=505 nm) and red (λ_max=650 nm) luminescence of Gd(hfac)₃, Gd(tta)₃ and Gd(qu)₃, respectively, originates from the lowest-energy intraligand triplets.     

Oxidation of malononitrile complex of pentaammineruthenium(II)

The oxidation of Ru(NH₃)₅NCCH₂CN²⁺ complex by the peroxydisulfate ion in the aqueous solution yields two products, Ru(NH₃)₅NHCOCH₂CN²⁺ with λ_max = 373 nm and withλ_max = 863 nm. The distribution of the products and the amount of oxidant required for the maximum yield of the binuclear complex depend on the acidity of the solution. The production of the binuclear complex was favored at lower acid concentrations. The formation of CC bond in the binuclear complex was characterized by both the spectral and the electrochemical results. A mechanism for the oxidation has been proposed by the kinetic studies in the region of 0.001-0.10 M acid concentrations.     

Probing the structural determinants of yellow fluorescence of a protein from Phialidium sp

Fluorescent proteins homologous to green fluorescent protein (avGFP) display pronounced spectral variability due to different chromophore structures and variable chromophore interactions with the surrounding amino acids. To gain insight into the structural basis for yellow emission, the 3D structure of phiYFP (λ_em = 537 nm), a protein from the sea medusa Phialidium sp., was built by a combined homology modeling – mass spectrometry approach. Mass spectrometry of the isolated chromophore-bearing peptide reveals that the chromophore of phiYFP is chemically identical to that of avGFP (λ_em = 508 nm). The experimentally acquired chromophore structure was combined with the homology-based model of phiYFP, and the proposed 3D structure was used as a starting point for identification of the structural features responsible for yellow fluorescence. Mutagenesis of residues in the local chromophore environment of phiYFP suggests that multiple factors cooperate to establish the longest-wavelength emission maximum among fluorescent proteins with an unmodified GFP-like chromophore.     

Investigation into the mechanism of λmax shifts and their dependence on pH for the 2-hydrazinopyridine derivatives of two copper amine oxidases

Copper amine oxidases (CuAO), from Escherichia coli (ECAO) and pea seedling (PSAO) were reacted with an excess of the hydrazine derivative 2-hydrazinopyridine (2HP) to form an initial, strongly absorbing adduct, (adduct 1; λ_max 420–430 nm) formed by the covalent binding of 2HP with the active site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ). Thermal incubation of buffered solutions of adduct 1 (pH 5.65–10.7) or addition of KOH solution (giving a final pH of 13–15) led isosbestically to a dramatic λ_max shift yielding adduct 2 (λ_max 520–530 nm). For both ECAO and PSAO, an increase in pH resulted in increased formation of adduct 2 with concomitant loss of adduct 1. Maximum adduct 2 formation occurred at pH 9.84 in ECAO and at pH 10.7 in PSAO. Beyond these pH levels, adduct 2 formation occurred to a much lesser extent which was independent of pH, suggesting enzyme denaturation. It is proposed that the conversion of adduct 1 to adduct 2 occurs as a result of hydrazone to azo conversion mediated by loss of a single proton, possibly to the active site base. It is further postulated that adduct formation and subsequent deprotonation can be likened to the substrate and product Schiff base complexes in the reductive half cycle of copper/TPQ containing amine oxidases. As part of this study an extinction coefficient at 280 nm was determined for ECAO by gravimetric analysis. This yielded a value of 2.1×10⁵ M⁻¹ cm⁻¹ giving rise to the need of a correction factor when estimating the protein concentration from an absorbance reading at 280 nm. Using the estimated molecular mass of 160 kDa for the homodimeric ECAO, a correction factor of 0.76 must be applied.     

Is the purple color of bacteriorhodopsin maintained by lipid-protein interactions?

When bacteriorhodopsin is delipidated and purified in detergents, its purple chromophore can be reversibly titrated to a red one. The pK_a of this equilibrium depends on the nature of the detergent in which bacteriorhodopsin is dispersed. In the absence of solvating amphiphiles, lipid-free detergent-free bacteriorhodopsin is red (λ_max = 480 nm) at pH higher than 3.5.     

Photoredox reactivity of copper(II)-3,5-diisopropylsalicylate induced by ligand-to-metal charge transfer excitation of the copper-phenolate chromophore

The electronic spectrum of Cu^II(dps)₂ in CH₃CN with dps=3,5-diisopropylsalicylate shows a ligand field absorption at λ_max=711 nm (ε=140 M⁻¹ cm⁻¹), and a phenolate to Cu(II) ligand-to-metal charge transfer (LMCT) band at λ_max=428 nm (ε=950). LMCT excitation of Cu^II(dps)₂ leads to the reduction of Cu(II) to Cu(I). Copper(II) disappears with φ=2.8×10⁻³ at λ_irr=436 nm.     

Structure and siderophore activity of ferric schizokinen

Schizokinen, a citrate-containing dihydroxamate, is a siderophore produced by Bacillus megaterium and Anabaena sp. The involvement of the citrate α-hydroxycarboxylate moiety in iron chelation was investigated by comparing the iron binding behavior of schizokinen with that of acetylschizokinen, a derivative in which the citrate hydroxyl group was modified by acetylation. Ferric schizokinen was found to exhibit an absorption spectrum (λ_max = 460 nm) characteristic of a dihydroxamate below pH 2.5, with an isosbestic shift to a citrate dihydroxamate spectrum (λ_max = 395 nm) above pH 4. Ferric acetylschizokinen also had a dihydroxamate absorption spectrum (λ_max = 465 nm) at low pH. However, its spectral shift (λ_max = 420 nm) and intensity above pH 4 were more typical of a ferric trihydroxamate. The molecular weight and electrophoretic mobility of ferric acetylschizokinen are consistent with a dimeric Fe₂ (acetylschizokinen)₃ structure, whereas ferric schizokinen appears to exist as a monomeric 1:1 complex Despite the differences in molecular weight and α-hydroxycarboxylate coordination, both complexes are effective in promoting iron uptake in Anabaena.     

Iodine binding property of a ternary complex consisting of starch,protein, and free fatty acids

A ternary complex consisting of amylose, whey protein, and free fatty acids (FFA) has been identified in our previous investigations, and its iodine binding properties were investigated. After reaction with iodine solution, an absorption peak (λ_max) at 620 nm was shown for pure amylose whereas the λ_max decreased to 510 nm when amylose was first complexed with FFA. Interestingly, a λ_max of 550 nm with an intermediate absorbance was observed for the ternary complex indicating its intermediate spectrophotometric property. Consistently, the amount of iodine bound by the ternary complex was between free amylose and typical amylose–FFA complex from potentiometric titration indicating the amylose–FFA complex within the ternary complex is less compact and more space is left for iodine binding. This in-between property of the ternary complex suggests it can be used as a molecular carrier to accommodate a forth component in addition to its functional lipids carrying capability in food product development.     

A circular dichroic absorption study of the reaction of oxidized pyridine nucleotides with cyanide ions.

A Circular Dichroic absorption study of the reaction of oxidized pyridine nucleotides with cyanide ions fully confirms the occurence of a very weak Cotton effect around 435 nm in the Circular Dichroic spectrum of the reduced coenzymes and therefore the very faint transition (λ_max = 435 nm; ?_max ～ 1 M^?1 cm^?1) from which the Cotton effect originates.     

The redox potentials of the b-type cytochromes of higher plant chloroplasts

1. In fresh chloroplasts, three b-type cytochromes exist. These are b-559_HP (λ_max, 559 nm; E_m at pH 7, +370 mV; pH-independent E_m), b-559_LP (λ_max, 559 nm; E_m at pH 7, +20 mV; pH-independent E_m) and b-563 (λ_max, 563 nm; E_m at pH 7, ?110 mV; pH-independent E_m). b-559_HP may be converted to a lower potential form (λ_max, 559 nm; E_m at pH 7, +110 mV; pH-independent E_m).2. In catalytically active b-f particle preparations, three cytochromes exist. These are cytochrome f (λ_max, 554 nm; E_m at pH 7, +375 mV, pK on oxidised cytochrome at pH 9), b-563 (λ_max, 563 nm; E_m at pH 7, ?90 mV, small pH-dependence of E_m) and a b-559 species (λ_max, 559 nm, E_m at pH 7, +85 mV; pH-independent E_m).3. A positive method of demonstration and estimation of b-559_LP in fresh chloroplasts is described which involves the use of menadiol as a selective reductant of b-559_LP.     

Multiple cytochromes b in Mycobacterium phlei

Electron transport particles from $\text{M. phlei}$ contain at least 3 different active forms of cytochrome b, one reduced by NADH, with a λ_max at 563 nm (b_N563), and the other two reduced by either succinate or NADH, with λ_max at 559 and 563 nm (b_S559) and (b_S563). Low temperature λ_max for cytochrome b reduction with NADH or succinate are described. During steady state only b_S563 was observed with succinate. In the presence of ATP, succinate reduced an increased amount of a b563. A branching of the NAD⁺-linked pathway and a convergence at the level of cytochrome c is suggested, with only one branch accessible to succinate.     

Electroretinogram and the spectral sensitivity of the compound eyes in the firefly Photuris versicolor (Coleoptera-Lampyridae): A correspondence between green sensitivity and species bioluminescence emission

ERGs were recorded from the dorsal sector of dark- and chromatic-adapted compound eyes in the dark-active firefly Photuris versicolor ♀ and ♂ at different wavelengths across the spectrum ranging from 320 nm to 700 nm over 4.5 log units of change in the stimulus intensity. ERG elicited by white light stimulus was an on-negative monophasic wave typical of scotopic eyes. ERGs elicited by chromatic stimuli differed in their waveform characteristics in the short (near-u.v. and violet) and long (green-yellow) wavelengths. The slope of the intensity-response curves at different stimulus wavelengths were similar for phasic response and differed for the plateau component of the ERG. The spectral sensitivity curves obtained under dark- and chromatic-adapted conditions revealed peaks in the near-u.v. (λ_max, 380 nm) and in the green (λ_max 550 nm), suggesting the presence of at least two receptor types in the dorsal sector of the compound eyes of P. versicolor. The green (550 nm) peak corresponds with the species bioluminescence emission peak (552 nm).