Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin

The preparations of N alpha-Fmoc-3-nitro-L-tyrosine and N-Boc-anthranilic acid Dhbt ester and their application to parallel multiple column solid-phase peptide synthesis is described. A series of peptide substrates containing an anthraniloyl group at the amino terminus and a 3-nitrotyrosyl residue close to the carboxyl terminus have been synthesized. The fluorescence of the anthraniloyl group, intramolecularly quenched by the 3-nitrotyrosine, increases with cleavage of peptide bonds situated between the two groups. The quenching mechanism is of the long-range resonance energy transfer type and long peptide substrates were constructed and used for kinetic measurement on subtilisin Carlsberg and pepsin. Complete quenching was observed even with more than 20 A between the centers of the chromophores, and substrates with up to 50 A between the chromophores were synthesized. The importance of long substrates for optimal enzymatic activity was demonstrated.     

A method of screening artificial substrates for proteolytic enzymes

A method of screening of proteolytic enzyme's substrates is proposed. An equimolar mixture of substrates consisting of peptide and easily detectable chromophore moieties (all chromophores in the mixture must be different) is subjected to enzymatic treatment. The cleaved chromophore groups, which are products of the substrate proteolysis, are quantitatively determined by chromatography. The Kcat/Km ratio is greater for substrates with higher initial rate accumulation of proteolysis products. The method is illustrated by screening of peptide derivatives of aminonaphtalene sulphonamides for trypsin assay. Proteolysis products are determined by HPLC with absorption detection or by TLC with fluorescence detection.     

Ciliary dynein conformational changes as evidenced by the extrinsic fluorescent probe 8-anilino-1-naphthalenesulfonate

The binding of 8-anilino-1-naphthalenesulfonate (ANS) to ciliary dynein ATPase leads to a marked increase in the dye's fluorescence intensity, accompanied by a blue shift in the observed fluorescence emission maximum. We found that dynein has 37 +/- 3 ANS binding sites and that experimentally applied ANS concentrations failed to alter enzyme activity. The fluorescence properties of the enzyme-dye complex were used to learn more about the binding characteristics of dynein substrates and effectors and to probe for possible conformational changes of the enzyme. The fluorescence of the dynein-ANS complex is increased by a number of substrates, including ATP, GTP, and UTP. The transfer of excitation energy from dynein chromophores to adsorbed ANS was also investigated. Our findings indicate that dynein appears to undergo a localized conformational change in its interaction with ATP. Native dynein was also found to be conformationally different from heat-activated or NEM-modified enzyme as evidenced by the emission and excitation spectra of the various enzyme-ANS complexes.     

Synthesis and evaluation of fluorescent probes for the detection of calpain activity

Two new probes for the detection of calpain I activity based on fluorescence resonance energy transfer technology have been synthesized and evaluated. The probes incorporated the cleavage site present in alpha-spectrin, a naturally occurring substrate of calpain I. The design of the internally quenched substrates is such that the calpain-sensitive bond of the peptides (between the Tyr-Gly residues) is located centrally between the donor and the quencher chromophores. The calpain assay protocol is capable of detecting enzymatic activity in the nanomolar region.     

Water-soluble rylene dyes as high-performance colorants for the staining of cells

The synthesis of perylene and terrylene chromophores carrying a single poly(ethylene oxide) chain is presented. These chromophores reveal a strong solvatochromic behavior: High fluorescence in nonpolar solvents and weak fluorescence in polar solvents which is mainly attributed to aggregation. Therefore, such chromophores are attractive candidates as sensitive fluorescent probes reflecting the polarity of their environment. In particular, their suitability for the staining of cellular membranes is presented in detail.     

Heterotrimeric collagen peptides as fluorogenic collagenase substrates: synthesis, conformational properties, and enzymatic digestion

The collagenase cleavage site of collagen type I, i.e., the sequence portions 772-784 (P(4)-P(9)') and 772-785 (P(4)-P(10)') of the two alpha1-chains and the sequence portion 772-784 (P(4)-P(9)') of the alpha2-chain, were assembled in an alpha1alpha2alpha1' register by C-terminal cross-linking of these peptides with an artificial cystine knot. The triple-helical conformation of the construct was stabilized by N-terminal extensions with (Gly-Pro-Hyp)(5) repeats. The gaps in the sequence alignment were filled up, and the alpha1-chain was dansylated and the alpha1'-chain was acylated with a tryptophan residue to place in spatial proximity the two chromophores for an efficient fluorescence resonance energy transfer. Although the incorporation of the two N-terminal chromophores leads to partial destabilization of the overall triple-helical fold, the heterotrimer behaved as a collagen-like substrate of the matrix metalloproteinases MMP-1 and MMP-13. Cleavage of the fluorogenic heterotrimer leads to a 6-fold increase in fluorescence intensity, thus making it a useful fluorogenic substrate for interstitial collagenases. With this folded heterotrimeric collagen molecule it was shown that fluorescence resonance energy transfer, as applied so far only for the design of linear fluorogenic enzyme substrates, can also be exploited in conformation dependency.     

Development of detection system of extraterrestrial microorganisms]

A noble method for the exploration of terrestrial and extraterrestrial soil microorganisms, especially targeted for Mars, has been developed. The method is based on the microscopic observation using fluorescence techniques. Microorganisms could be fluorescent by adsorption, enzymatic cleavage of extrinsic fluorescence chromophores such as acridine orange, ANS and SFDA, and also by intrinsic chromophores. The characteristic points of our fluorescence method are shown below. 1. The present method detected all the culturable cells tested (about 200 species from bacteria to eukaryofic cells). 2. Microorganisms in soil were much brighter than background fluorescence of soil. Cell shapes and location were clearly observed. 3. An esterase substatum SFDA, discriminated vital (reproductive) cells from dead. On the other hand, a membrane probe, ANS, detected both vital and dead cells. 3. Pre-treatment of cells with bleaching reagents improved the detection efficiency. Especially, this pretreatment was effecfive in Fungi with black chromophores. 4. Some anaerobic microorganisms such as methanogenic bacteria with intrinsic chromophores can be detected without stain. 5. Application of the technique to terrestrial soil revealed that more than 100 times larger cell density was obtained compared to the value obtained by the classic plate counting technique. Vertical distribution of microorganism of soil microorganisms from Mt. Shigayama showed that, at surface, cell density was small and maximum was shown below 15 cm from surface. 6. Some pre-biotic cell (cell like aggregates composed of amino acids) could be detected by SFDA or ANS. It can be concluded that the fluorescence technique is one of the most promising method for the exploration of extraterrestrial microorganisms.     

A general fluorescence-based coupled assay for S-adenosylmethionine-dependent methyltransferases

We have developed a simple and sensitive fluorescence-based two-step coupled enzyme assay to report the activity of S-adenosylmethionine-dependent methyltransferases. This assay relies on a fluorescein-cystamine-methyl red (FL-S-S-MR) reporter molecule that can be activated by thiols. In the absence of thiols, fluorescence from the reporter is quenched through fluorescence resonance energy transfer between the two chromophores. In this report, we use catechol-O-methyltransferase with the addition of S-adenosylhomocysteine hydrolase to produce the thiol homocysteine. The presence of homocysteine leads to disulfide bond cleavage in the cystamine tether and fluorescence dequenching as the uncoupled chromophores are diluted into the surrounding media. The sensitivity and specificity of FL-S-S-MR to thiols enabled detection of 相似文献   

Unfolding of C-phycocyanin followed by loss of non-covalent chromophore-protein interactions 1. Equilibrium experiments

Optical spectroscopic properties of the covalently linked chromophores of biliproteins are profoundly influenced by the state of the protein. This has been used to monitor the urea-induced denaturation of C-phycocyanin (CPC) from Mastigocladus laminosus and its subunits. Under equilibrium conditions, absorption, fluorescence and circular dichroism of the chromophores were monitored, as well as the circular dichroism of the polypeptide. Treatment of CPC trimers (alphabeta)3 resulted first in monomerization (alphabeta), which was followed by a complex unfolding process of the protein. Loss of chromophore fluorescence is the next process at increasing urea concentrations; it indicates increased flexibility of the chromophore while maintaining the native, extended conformation, and a less compact but still native-like packing of the protein in the regions sampled by the chromophores. This was followed by relaxation of the chromophores from the energetically unfavorable extended to a cyclic-helical conformation, as reported by absorption and CD in the visible range, indicating local loss of protein structure. Only then is the protein secondary structure lost, as reported by the far-UV CD. Sequential processes were also seen in the subunits, where again the chromophore-protein interactions were reduced before the unfolding of the protein. It is concluded that the bilin chromophores are intrinsic probes suitable to differentiate among different processes involved in protein denaturation.     

Effect of glycerol and PVA on the conformation of photosystem I

Single-molecule spectroscopy at cryogenic temperatures was used to examine the impact of buffer solution, glycerol/buffer mixtures (25% and 66%), and poly(vinyl alcohol) (PVA) films on the conformation of photosystem I (PSI) from Thermosynechoccocus elongatus. PSI holds a number of chromophores embedded at different places within the protein complex that show distinguishable fluorescence at low temperatures. The fluorescence emission from individual complexes shows inter- and intracomplex heterogeneity depending on the solution wherein PSI was dissolved. Statistical evaluation of spectra of a large number of complexes shows that the fluorescence emission of some of these chromophores can be used as sensors for their local nanoenvironment and some as probe for the conformation of the whole protein complex. Preparation in glycerol/buffer mixtures yields a high homogeneity for all chromophores, indicating a more compact protein conformation with less structural variability. In buffer solution a distinct heterogeneity of the chromophores is observed. PSI complexes in PVA show highly heterogeneous spectra as well as a remarkable blue shift of the fluorescence emission, indicating a destabilization of the protein complex. Photosystem I prepared in PVA cannot be considered fully functional, and conclusions drawn from experiments with PSI in PVA films are of questionable value.     

Single-molecule spectroscopy selectively probes donor and acceptor chromophores in the phycobiliprotein allophycocyanin

We report on single-molecule fluorescence measurements performed on the phycobiliprotein allophycocyanin (APC). Our data support the presence of a unidirectional F?rster-type energy transfer process involving spectrally different chromophores, alpha84 (donor) and beta84 (acceptor), as well as of energy hopping amongst beta84 chromophores. Single-molecule fluorescence spectra recorded from individual immobilized APC proteins indicate the presence of a red-emitting chromophore with emission peaking at 660 nm, which we connect with beta84, and a species with the emission peak blue shifted at 630 nm, which we attribute to alpha84. Polarization data from single APC trimers point to the presence of three consecutive red emitters, suggesting energy hopping amongst beta84 chromophores. Based on the single-molecule fluorescence spectra and assuming that emission at the ensemble level in solution comes mainly from the acceptor chromophore, we were able to resolve the individual absorption and emission spectra of the alpha84 and beta84 chromophores in APC.     

Unfolding of C-phycocyanin followed by loss of non-covalent chromophore-protein interactions: 1. Equilibrium experiments

Optical spectroscopic properties of the covalently linked chromophores of biliproteins are profoundly influenced by the state of the protein. This has been used to monitor the urea-induced denaturation of C-phycocyanin (CPC) from Mastigocladus laminosus and its subunits. Under equilibrium conditions, absorption, fluorescence and circular dichroism of the chromophores were monitored, as well as the circular dichroism of the polypeptide. Treatment of CPC trimers (αβ)₃ resulted first in monomerization (αβ), which was followed by a complex unfolding process of the protein. Loss of chromophore fluorescence is the next process at increasing urea concentrations; it indicates increased flexibility of the chromophore while maintaining the native, extended conformation, and a less compact but still native-like packing of the protein in the regions sampled by the chromophores. This was followed by relaxation of the chromophores from the energetically unfavorable extended to a cyclic-helical conformation, as reported by absorption and CD in the visible range, indicating local loss of protein structure. Only then is the protein secondary structure lost, as reported by the far-UV CD. Sequential processes were also seen in the subunits, where again the chromophore-protein interactions were reduced before the unfolding of the protein. It is concluded that the bilin chromophores are intrinsic probes suitable to differentiate among different processes involved in protein denaturation.     

Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic properties.

Heterodimeric dyes are described which bind tightly to double-stranded (dsDNA) with large fluorescence enhancements. These dyes are designed to exploit energy transfer between donor and acceptor chromophores to tune the separation between excitation and emission wavelengths. The dyes described here absorb strongly at the 488 nm argon ion line, but emit at different wavelengths, and can be applied to multiplex detection of various targets. The chromophores in these dyes, a thiazole orange-thiazole blue heterodimer (TOTAB), two different thiazole orange-ethidium heterodimers (TOED1 and TOED2), and a fluorescein-ethidium heterodimer (FED), are in each case linked through polymethyleneamine linkers. The emission maxima of the DNA-bound dyes lie at 662 (TOTAB), 614 (TOED 2), and 610 nm (FED). The dyes showed a > 100 fold enhancement of the acceptor chromophore fluorescence on binding to dsDNA and no sequence selectivity. In comparison with direct 488 nm excitation of the constituent monomeric dyes, in the heterodimers the fluorescence of the acceptor chromophores was greatly enhanced and the emission of the donor chromophores quenched by over 90%. The acceptor emission per DNA-bound dye molecule was constant from 100 DNA bp:dye to 20 bp:dye and decreased sharply at higher dye:DNA ratios.     

Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores.

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.     

The microenvironment around the chromophores and its changes due to the association states in C-phycocyanin isolated from the cyanobacterium Mastigocladus laminosus

The chromophore-protein interaction in C-phycocyanin was investigated as a function of the association state of the protein. Changes in the microenvironment around the chromophores were monitored by the following three indices: (1) the accessibility of a small molecule to the chromophore; (2) the fluorescence from aromatic amino acid residues; and (3) the effect of configurational changes of the chromophore on the conformation of the polypeptide chain. In the C-phycocyanin trimer, all the chromophores are shielded from the aqueous phase, probably by contact between subunits, and by a loop structure which surrounds the chromophores, even though that loop structure is not shown by X-ray analysis (Schirmer, T., Bode, W., Huber, R., Sidler, W. and Zuber, H. (1985) J. Mol. Biol. 184, 257–277). The polypeptide folding depends on the electronic structure of the chromophores; the oxidized chromophore of the α subunit inhibits the formation of the trimer and the reduced state of the chromophore of the α subunit allows the formation of trimers, in which the chromophores have the same electronic structure as in the monomers. The fluorescence from the aromatic amino acid residues showed that the conformational changes were induced by the reduced chromophore. These results indicate that the chromophore structure and the protein conformation affect each other. A definite configuration of the chromophore and also a definite conformation of the polypeptide are necessary for the intact energy transfer within C-phycocyanin.     

Allophycocyanin complexes of the phycobilisome from Mastigocladus laminosus. Influence of the linker polypeptide L8.9C on the spectral properties of the phycobiliprotein subunits

The following phycobiliproteins and complexes of the allophycocyanin core were isolated from phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus: alpha AP, beta AP, (alpha AP beta AP), (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C, (alpha APB alpha AP2 beta AP3)L8.9C. The six proteins and complexes were characterised spectroscopically with respect to absorption, oscillator strength, extinction coefficient, fluorescence emission, relative quantum yield, fluorescence emission polarisation and fluorescence excitation polarisation. The interpretation of the spectral data was based on the three-dimensional structure model of (alpha PC beta PC)3 (Schirmer et al. (1985) J. Mol. Biol. 184, 257-277), which is related to the allophycocyanin trimer. The absorption and CD spectra of the complexes (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C and (alpha APB alpha AP2 beta AP3)L8.9C could be deconvoluted into the spectra of the phycobiliprotein subunits. The assumptions made for the deconvolution could be checked by the synthesis of the spectra of (alpha APB beta AP)3. The synthesised spectra are in good agreement with the corresponding measured spectra published by other authors. Considering the deconvoluted spectra the following influences on the chromophores could be ascribed to L8.9C: L8.9C neither influences the alpha AP nor the alpha APB chromophores. L8.9C shifts the absorption maximum of the beta AP chromophore to longer wavelength than the absorption maximum of the alpha AP chromophore in trimeric complexes. L8.9C increases the oszillator strength of the beta AP chromophores to about the value of the alpha AP chromophores in trimeric complexes. L8.9C turns the beta AP chromophores from sensitizing into weak fluorescing chromophores. By means of the hydropathy plot and the predicted secondary structure, a postulated three-fold symmetry in the tertiary structure of L8.9C could be confirmed.     

Origin of the 701-nm fluorescence emission of the Lhca2 subunit of higher plant photosystem I

Photosystem I of higher plants is characterized by red-shifted spectral forms deriving from chlorophyll chromophores. Each of the four Lhca1 to -4 subunits exhibits a specific fluorescence emission spectrum, peaking at 688, 701, 725, and 733 nm, respectively. Recent analysis revealed the role of chlorophyll-chlorophyll interactions of the red forms in Lhca3 and Lhca4, whereas the basis for the fluorescence emission at 701 nm in Lhca2 is not yet clear. We report a detailed characterization of the Lhca2 subunit using molecular biology, biochemistry, and spectroscopy and show that the 701-nm emission form originates from a broad absorption band at 690 nm. Spectroscopy on recombinant mutant proteins assesses that this band represents the low energy form of an excitonic interaction involving two chlorophyll a molecules bound to sites A5 and B5, the same protein domains previously identified for Lhca3 and Lhca4. The resulting emission is, however, substantially shifted to higher energies. These results are discussed on the basis of the structural information that recently became available from x-ray crystallography (Ben Shem, A., Frolow, F., and Nelson, N. (2003) Nature 426, 630-635). We suggest that, within the Lhca subfamily, spectroscopic properties of chromophores are modulated by the strength of the excitonic coupling between the chromophores A5 and B5, thus yielding fluorescence emission spanning a large wavelength interval. It is concluded that the interchromophore distance rather than the transition energy of the individual chromophores or the orientation of transition vectors represents the critical factor in determining the excitonic coupling in Lhca pigment-protein complexes.     

DNA-templated formation and luminescence of diphenylacetylene dimeric and trimeric complexes

We report on spectral features for two and three diphenylacetylene chromophores aligned in close proximity in aqueous solution by self assembly of attached oligonucleotide arms. Two duplex systems were examined in detail. One was formed by hybridization (Watson-Crick base pairing) of two oligonucleotide 10-mers, each containing the diphenylacetylene insert. The other was generated by self-folding of a 36-mer oligonucleotide containing two diphenylacetylene inserts. The triplex system was obtained by hybridization (Hoogsteen base pairing) of a 16-mer oligonucleotide diphenylacetylene conjugate to the folded 36-mer hairpin. Formation of duplex and triplex entities from these conjugates was demonstrated experimentally by thermal dissociation and spectroscopic studies. The UV and CD spectra for the duplex systems exhibit bands in the 300-350 nm region attributable to exciton coupling between the two chromophores, and the emission spectra show a strong band centered at 410 nm assigned to excimer fluorescence. Addition of the third strand to the hairpin duplex has little effect on the CD spectrum in the 300-350 nm region, but leads to a negative band at short wavelengths characteristic of a triplex and to a strongly enhanced band at 410 nm in the fluorescence spectrum. The third strand alone shows a broad fluorescence band at approximately 345-365 nm, but this band is virtually absent in the triplex system. A model for the triplex system is proposed in which two of the three aligned diphenylacetylenes function as a ground state dimer that on excitation gives rise to the exciton coupling observed in the UV and CD spectra and to the excimer emission observed in the fluorescence spectrum. Excitation of the third chromophore results in enhanced excimer fluorescence, as a consequence of energy transfer from the locally excited singlet of one chromophore to the ground state dimer formed by the other two chromophores.     

Deconvolution of C-phycocyanin beta-84 and beta-155 chromophore absorption and fluorescence spectra of cyanobacterium Mastigocladus laminosus.

Absorption and fluorescence spectra of the C-phycocyanin beta-subunit were quantitatively deconvoluted into component spectra of the beta-84 and beta-155 chromophores. The deconvolution procedure was based on a theoretical treatment of polarization properties. Four kinds of spectra (absorption, emission, emission polarization, and excitation polarization) measured on C-phycocyanin isolated from the cyanobacterium Mastigocladus laminosus were used as the experimental data set. Without any assumption of spectral shape, the absorption and fluorescence spectra of both chromophores were unambiguously resolved and their fluorescence quantum yields were evaluated. By combining the spectra of the alpha-subunit, independently measured, with the resolved spectra of the beta-subunit, the fluorescence and fluorescence polarization spectra and the fluorescence quantum yield of the monomer were estimated; they agree with experimental values to within an acceptable error. Further, the matrix of energy transfer rates in the monomer was estimated; it gave a significantly different result (by up to 40%) from previously estimated ones.     

Three-stage refolding/unfolding of the dual-color beta-subunit in R-phycocyanin from Polysiphonia urceolata

The conformational changes during refolding and unfolding of the dual-color beta-subunit in R-phycocyanin (R-PC) were monitored by the spectra, fluorescence anisotropy, and FRET. It was observed that both of the refolding and unfolding of the beta-subunit would undergo a three-stage conformational change, but in a reverse order. During the refolding process, at the first stage, the configuration of the tetrapyrrole chromophores transformed from the cyclohelical to the extended one, suggested by the blue-shifted spectra. At the second stage, recovery of the hydrogen-bond and hydrophobic interaction network fixed the chromophore in a more rigid configuration, suggested by a linear increase in the total fluorescence yield. At the third stage, the increase of the FRET efficiency suggested a protein-framework movement that made the two chromophores closer or/and into a more parallel orientation. The fluorescence anisotropy further confirmed the three-stage model.