Factors affecting the estimation of the relative amount of chromophore and chromophore area by the two-wavelength method of patau and ornstein

Summary Factors influencing the calculation of the relative amount of chromophore and the chromophore area by the two-wavelength method are examined. The study was carried out with the help of models and further tested on Feulgen stained preparations. Except for certain restrictions the difference between the chromophore area as calculated from the two transmissions measurements and the chromophore area obtained by planimetry can be used as a guide for determining the proper measuring conditions, including the choise of the two wavelengths.     

Neocarzinostatin chromophore binds to deoxyribonucleic acid by intercalation

The nonprotein chromophore of neocarzinostatin was found to share many of the characteristics of classical intercalators in its interaction with DNA. Viscosity studies with PM2 DNA indicated that the DNA helix unwinding induced by the chromophore was 0.82 times that of ethidium or 21 degrees. Electric dichroism of the chromophore--DNA complex showed that each bound chromophore molecule lengthened DNA by 3.3 A and that absorbance transitions of the chromophore at 315--385 nm were oriented approximately parallel to DNA bases, as expected for an intercalated aromatic ring. Binding to DNA induced strong hypochromicity and a pronounced red shift in the absorbance spectrum of the chromophore. Spectrophotometric titrations suggested at least two types of chromophore binding sites on DNA; one type of site was saturated at rb = 0.125 chromophore molecule/nucleotide, but binding to additional sites continued to at least rb = 0.3. These physical--chemical studies were performed at pH 4--5 in order to keep the chromophore stable, but chromophore bound to an excess of DNA at pH 7 showed a stable absorbance spectrum identical with that seen at pH 4--5, suggesting that a similar type of binding occurs at neutral pH. Chromophore which had spontaneously degraded in pH 8 buffer did not bind to DNA at all, as judged by absorbance spectroscopy. The degree of protection afforded by DNA against spontaneous chromophore degradation implied a dissociation constant of approximately 5 microM for the DNA--chromophore complex at neutral pH and physiological ionic strength. Supercoiled DNA was nearly twice as effective as relaxed DNA in protecting chromophore from degradation, providing additional evidence for intercalation at neutral pH. Comparison of absorbance, fluorescence, and dichroism spectra suggests that the naphthalene ring system is the intercalating moiety.     

Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins

The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein we report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein "toolkit".     

Role of bulk water in hydrolysis of the rhodopsin chromophore

Rhodopsin (Rho) is a prototypical G protein-coupled receptor that changes from an inactive conformational state to a G protein-activating state as a consequence of its retinal chromophore isomerization, 11-cis-retinal → all-trans-retinal. The photoisomerized chromophore covalently linked to Lys(296) by a Schiff base is subsequently hydrolyzed, but little is known about this reaction. Recent research indicates a significant role for tightly bound transmembrane water molecules in the Rho activation process. Atomic structures of Rho and hydroxyl radical footprinting reveal ordered waters within Rho transmembrane helices that are located close to highly conserved and functionally important receptor residues, forming a hydrogen bond network. Using (18)O-labeled H(2)O, we now report that water from bulk solvent, but not tightly bound water, is involved in the hydrolytic release of chromophore upon Rho activation by light. Moreover, small molecules (and presumably, water) enter the Rho structure from the cytoplasmic side of the membrane. Thus, this work indicates two distinct origins of water vital for Rho function.     

Structure of the chromophore fragment of copsomycin

The chromophore moiety of the copsomycin molecule was isolated in the form of dimethyl ether as an individual substance from the products of antibiotic acid hydrolysis. Physicochemical properties of the chromophore were studied. UV, 1H and 13C NMR, mass spectrometry and elementary analysis showed that the copsomycin molecule moiety was identical to the chromophore moiety of the molecule of multiomycin or nosiheptide.     

Binding of the nonprotein chromophore of neocarzinostatin to deoxyribonucleic acid

The methanol-extracted, nonprotein chromophore of neocarzinostatin (NCS), which has DNA-degrading activity comparable to that of the native antibiotic, was found to have a strong affinity for DNA. Binding of chromophore was shown by (1) quenching by DNA of the 440-nm fluorescence and shifting of the emission peak to 420 nm, (2) protection by DNA against spontaneous loss of activity in aqueous solution, and (3) inhibition by DNA of the spontaneous generation of 490-nm fluorescence. Good quantitative correlation was found between these three methods in measuring chromophore binding. There was nearly a 1:1 correspondence between loss of chromophore activity and generation of 490-nm fluorescence, suggesting spontaneous degradation of active chromophore to a highly fluorescent product. Chromophore showed a preference for DNA high in adenine + thymine content in both fluorescence quenching and protection studies. NCS apoprotein, which is known to bind and protect active chromophore, quenched the 440-nm fluorescence, shifted the emission peak to 420 nm, and inhibited the generation of 490-nm fluorescence. Chromophore had a higher affinity for apoprotein than for DNA. Pretreatment of chromophore with 2-mercaptoethanol increased the 440-nm fluorescence seven-fold and eliminated the tendency to generate 490-nm fluorescence. The 440-nm fluorescence of this inactive material was also quenched by DNA and shifted to 420 nm, indicating an affinity for DNA comparable to that of untreated chromophore. However, its affinity for apoprotein was much lower than that of untreated chromophore. Both 2-mercapto-ethanol-treated and untreated chromophore unwound supercoiled pMB9 DNA, suggesting intercalation by both molecules. Since no physical evidence for interaction of native neocarzinostatin with DNA has been found, it is likely that dissociation of the chromophore from the protein and association with DNA are important steps in degradation of DNA by neocarzinostatin.     

Fungal phytochrome chromophore biosynthesis at mitochondria

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome‐dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme‐derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C‐terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000‐fold higher than the affinity of the holoprotein, suggesting a “kiss‐and‐go” mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.     

Chromophore selectivity in bacterial phytochromes: dissecting the process of chromophore attachment.

Bacterial phytochromes (Bphs) are ancestors of the well characterized plant photoreceptors. Whereas plant phytochromes perform their photoisomerization exclusively via a covalently bound bilin chromophore, Bphs are variable in their chromophore selection. This is demonstrated in the cyanobacterium Calothrix PCC7601 that expresses two Bphs, CphA and CphB. CphA binds phycocyanobilin (PCB) covalently, whereas CphB, lacking the covalently binding cysteine of the plant phytochromes, carries biliverdin IXalpha (BV) as the chromophore. Our experiments elucidate the different modes of chromophore-protein interaction in CphA and CphB and offer a rationale for their chromophore selectivity. The tight binding of BV by CphB prevents PCB from competing for the binding cavity. Even when the chromophore-binding cysteine has been inserted (CphB-mutant L266C), PCB replaces BV very slowly, indicating the tight, but not irreversible binding of BV. The mutant CphB L266C showed a redox-sensitivity with respect to its PCB binding mode: under reducing conditions, the chromoprotein assembly leads to spectra indicative for a covalent binding, whereas absence of dithiothreitol or its removal prior to assembly causes spectra indicative for noncovalent binding. Regarding the CphB-type Bphs lacking the covalently binding cysteine, our results support the involvement of the succeeding histidine residue in chromophore fixation via a Schiff base-like bond between the bilin A-ring carbonyl and the histidine imidazole group. The assembly process and the stability of the holo-proteins were strongly influenced by the concentration of added imidazole (mimicking the histidine side-chain), making the attachment of the chromophore via the histidine more likely than via another cysteine of the protein.     

The structure of the retinylidene chromophore in bathorhodopsin.

Resonance Raman data on bathorhodopsin (bovine and squid) at 95,77, and 4 degrees K support a mechanism of excitation proposed by Lewis in which both a protein conformational transition and chromophore structural alteration to a "dicisoid" configuration are required to generate the bathorhodopsin species observed in steady-state photostationary mixtures. However, these results also suggest that the molecular structure with a red-shifted chromophore absorption detected at room temperatures in 1 ps using picosecond absorption spectroscopy may not necessarily have the same chromophore conformation as the steady-state bathorhodopsin species.     

DNA photorepair: chromophore composition and function in two classes of DNA photolyases

DNA photolyase catalyzes the repair of pyrimidine dimers in UV-damaged DNA in a reaction which requires visible light. Class I photolyases (Escherichia coli, yeast) contain 1,5-dihydroFAD (FADH2) plus a pterin derivative (5,10-methenyltetrahydropteroylpolyglutamate). In class II photolyases (Streptomyces griseus, Scenedesmus acutus, Anacystis nidulans, Methanobacterium thermoautotrophicum) the pterin chromophore is replaced by an 8-hydroxy-5-deazaflavin derivative. The two classes of enzymes exhibit a high degree of amino acid sequence homology, suggesting similarities in protein structure. Action spectra studies show that both chromophores in each enzyme tested act as sensitizers in catalysis. Studies with E. coli photolyase show that the pterin chromophore is not required when FADH2 acts as the sensitizer but that FADH2 is required when the pterin chromophore acts as sensitizer. FADH2 is probably the chromophore that directly interacts with substrate in a reaction which may be initiated by electron transfer from the excited singlet state (1FADH2*) to form a flavin radical plus an unstable pyrimidine dimer radical. Pterin, the major chromophore in E. coli photolyase, may act as an antenna to harvest light energy which is then transferred to FADH2.     

The bleaching of the bacteriorhodopsin chromophore by ionizing radiation.

The visible chromophore of bacteriorhodopsin, BR(570), undergoes progressive bleaching when subjected to 60CO gamma-irradiation. The low G-value for bleaching confirms that the site of the chromophore is highly protected. Positive and negative circular dichroic (CD) bands associated with the chromosphone undergo concomitant decrease in a manner which is consistent with two independent chromophores rather than exciton coupling between neighbouring chromophoric site.     

Synthesis and properties of the chromophore of the asFP595 chromoprotein from Anemonia sulcata

A model compound for the chromophore within the purple nonfluorescent GFP-like chromoprotein asFP595 was synthesized. The postulated structure of the chromophore, 2-acetyl-4-(p-hydroxybenzylidene)-1-methyl-5-imidazolone, was taken from the high-resolution crystal structure analysis of intact asFP595 [Quillin, M. L., Anstrom, D., Shu, X., O'Leary, S., Kallio, K., Lukyanov, K. A., and Remington, S. J. (2005) Kindling Fluorescent Protein from Anemonia sulcata: Dark-State Structure at 1.38 A Resolution, Biochemistry 44, 5774-5787]. Erlenmeyer lactonization and oxidation of the methylene group attached to the heteroaromatic moiety with selenium dioxide were used at the key stages of the synthesis. The spectral properties of the model chromophore in solution and their dependence on the pH and polarity of the solvent were investigated. In water, the chromophore was found to exist in two forms, neutral and anionic, with a pK(a) of 7.1. In a dimethylformamide solution, the spectral properties of the anionic form closely match those of the native protein, demonstrating that under these conditions, the compound is an excellent model for the chromophore within native asFP595.     

Comparative studies of chromophore contents inside and outside the rhabdoms of arthropod compound eyes

Summary The intracellular distribution of visual pigments in the retinular cells of compound eyes of a butterfly (Papilio), a moth (Actias), a water scorpion (Ranatra), a dragonfly (Pantala), a crayfish (Procambarus), and a crab (Hemigrapsus) were investigated.The experiments measured the total surface area of rhabdom membrane in single compound eyes by light and electron microscopy, the densities of intramembrane particles in rhabdoms of compound eyes by freeze-fracture methods, and the quantities of chromophore molecules in compound eyes by high pressure liquid chromatography.Four species (moth, water scorpion, dragonfly, and crayfish) contain more than 75% of total chromophore probably as visual pigments in their rhabdoms, but the butterfly and the crab contain only 28.6% and 39.2%, respectively.The remainder of the chromophore molecules (Papilio 71.4%,Actias 5.7%,Ranatra 18.9%,Pantala 23.2%,Procambarus 24.3%, andHemigrapsus 60.8%) are supposed to exist mostly in the cytoplasm of the retinular cells. The localizing sites of such extrarhabdomeric chromophores are discussed in relation to the cytoplasmic membrane systems such as endoplasmic reticulum and lysosomal elements.In view of the results of the present experiment, it seems clear that the butterfly,Papilio, possesses a very large extra-rhabdomeric pool of chromophore that is available for rapid regeneration of visual pigment.Abbreviations HPLC high pressure liquid chromatography - ROR ratio of rhabdom to retinulae volume     

The gas-phase absorption spectrum of a neutral GFP model chromophore

We have studied the gas-phase absorption properties of the green fluorescent protein (GFP) chromophore in its neutral (protonated) charge state in a heavy-ion storage ring. To accomplish this we synthesized a new molecular chromophore with a charged NH(3) group attached to a neutral model chromophore of GFP. The gas-phase absorption cross section of this chromophore molecule as a function of the wavelength is compared to the well-known absorption profile of GFP. The chromophore has a maximum absorption at 415 +/- 5 nm. When corrected for the presence of the charged group attached to the GFP model chromophore, the unperturbed neutral chromophore is predicted to have an absorption maximum at 399 nm in vacuum. This is very close to the corresponding absorption peak of the protein at 397 nm. Together with previous data obtained with an anionic GFP model chromophore, the present data show that the absorption of GFP is primarily determined by intrinsic chromophore properties. In other words, there is strong experimental evidence that, in terms of absorption, the conditions in the hydrophobic interior of this protein are very close to those in vacuum.     

Solution structures of C-1027 apoprotein and its complex with the aromatized chromophore

C-1027 is one of the most potent antitumor antibiotic chromoproteins, and is a 1:1 complex of an enediyne chromophore having DNA-cleaving ability and a carrier apoprotein. The three-dimensional solution structures of the 110 residue (10.5 kDa) C-1027 apoprotein and its complex with the aromatized chromophore have been determined separately by homonuclear two-dimensional nuclear magnetic resonance methods. The apoprotein is mainly composed of three antiparallel beta-sheets: four-stranded beta-sheet (43-45, 52-54; 30-38; 92-94; 104-106), three-stranded beta-sheet (4-6; 17-22; 61-66), and two-stranded beta-sheet (70-72; 83-85). The overall structure of the apoprotein is very similar to those of other chromoprotein apoproteins, such as neocarzinostatin and kedarcidin. A hydrophobic pocket with approximate dimensions of 14 A x 12 A x 8 A is formed by the four-stranded beta-sheet and the three loops (39-42; 75-79; 97-100). The holoprotein (complex form with the aromatized chromophore) structure reveals that the aromatized chromophore is bound to the hydrophobic pocket found in the apoprotein. The benzodihydropentalene core of the chromophore is located in the center of the pocket and other substituents (beta-tyrosine, benzoxazine, and aminosugar moieties) are arranged around the core. Major binding interactions between the apoprotein and the chromophore are likely the hydrophobic contacts between the core of the chromophore and the hydrophobic side-chains of the pocket-forming residues, which is supplemented by salt bridges and/or hydrogen bonds. Based on the holoprotein structure, we propose possible mechanisms for the stabilization and the release of chromophore by the apoprotein.     

Accessibility of the iodopsin chromophore

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH₄ bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from isopsin in the iodopsin These facts suggests that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.     

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).     

Accessibility of the iodopsin chromophore.

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH4 bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from iodopsin in the dark. These facts suggest that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.     

Interaction of tyrosine residues with the chromophore in bacteriorhodopsin

We previously reported that the absorption spectrum at low temperatures of iodinated bacteriorhodopsin can be separated into four components with maxima at shorter wavelengths than in native bacteriorhodopsin. In this study, the time course of the formation of each spectral component after iodination was analyzed, revealing that these four components correspond to four different iodinated states of tyrosine residues interacting with the retinal chromophore of bacteriorhodopsin. Therefore at least two tyrosine residues interact with the chromophore of bacteriorhodopsin.     

On the linkages between chromophore and protein in biliproteins, VII. Amino acid sequence in the chromophore regions of C-phycoerythrin from Pseudanabaena W 1173 and Phormidium persicinum.

Bilipeptides from all chromophore regions were prepared by trypsin digestion of C-phycoerythrin from Pseudanabaena W 1173 and Phormidium persicinum. Analytical separation and quantitative determination of bilipeptides was achieved by isoelectric focusing, preparative isolation by gel chromatography and ion-exchange chromatography. Amino acid analysis revealed cysteine as the only amino acid common to all chromopeptides. Amino acid sequences were determined by Edman degradation and the dansyl-Edman technique. Sequences are different in all 5 and 6 chromophore regions, respectively. Possible homologies are discussed. A thioether linkage between ring A of the chromophore and cysteine was found in the bilipeptides (as before in biliproteins). A second linkage (serine ester) was found in only one peptide (CM 4.I from Pseudanabaena W 1173). This peptide absorbs as cation at a longer wavelength (559 nm) than the other bilipeptides (542 - 550 nm).