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1.
Vibrio fischeri strain Y-1 (ATCC 33715) emits light with a lambda max of 545 nm rather than the 485-nm emission typical of other strains of V. fischeri. The yellow emission is due to the interaction of the enzyme luciferase with a yellow fluorescent protein (YFP). On the basis of the N-terminal amino acid sequence of YFP, a mixed-sequence oligonucleotide probe was synthesized and used to isolate a 1.6-kbp HindIII fragment containing the first 208 bases of the gene that codes for YFP (luxY). Another synthetic oligonucleotide complementary to bases 167-184 of the YFP coding sequence was used to isolate a second (ca. 1.9 kbp) DNA fragment generated by digestion with both EcoRI and ClaI that contained the remainder of the luxY gene. The intact luxY gene, which encoded a 22,211-dalton polypeptide composed of 194 amino acid residues, was reconstructed from the two primary clones and is contained within a 765-bp SspI-XhoII fragment. Both strands of the entire luxY coding sequence were determined from the reconstructed gene, while the region surrounding the junction used in the reconstruction was also determined from the original partial clones. As with other genes that have been studied from V. fischeri, the luxY gene was unusually AT-rich. The sequence of luxY did not bear any apparent similarity to any of the sequences contained in the current GenBank database. Escherichia coli containing a plasmid with the luxY gene expresses a protein that reacts with antibody raised to authentic YFP.  相似文献   

2.
To elucidate the reversible change in the color of bioluminescence (BL) arising from Vibrio fischeri Y1, the relationship between the BL color and the redox state of endogenous yellow fluorescent protein (YFP), carrying riboflavin 5'-phosphate (FMN), has been investigated in vitro. YFP lost fluorescence with a maximum at 538 nm when reduced, and retrieved its original fluorescence upon reoxidation. Such a change in YFP fluorescence was analogous to that of free FMN. In the NADH/FMN oxidoreductase-coupled luciferase reaction with YFP, yellow BL peaking around 535 nm was largely depressed when sodium dithionite was added. This phenomenon can be attributed to the reduction of YFP; i.e., reduced YFP does not participate in the luciferase reaction as a secondary emitter. On admitting air into the reaction mixture, the yellow light characteristic of V. fischeri Y1 BL was regenerated. These results indicate that the reversible change in YFP fluorescence is caused by the redox change of YFP-bound FMN, and that the change in BL color between blue and yellow is associated with the redox state of YFP.  相似文献   

3.
4.
We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and (1)H, (13)C, and two-dimensional NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and fluorescence at 410 nm when excited at 343 nm. Analysis of the purified compound by reversed-phase HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure to be 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues, and a five-carbon ring. Because this cross-link contains two lysine residues and a dihydropyridinium ring, we assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests were made using a high-performance liquid chromatograph equipped with a diode array detector. These measurements revealed a significant enhancement of K2P in cataract lens proteins (613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of WS protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of water-soluble (WS) protein). These data provide chemical evidence for increased protein cross-linking during aging and cataract development in vivo. This new cross-link may serve as a quantitatively more significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.  相似文献   

5.
In previous studies involving Photobacterium species we proposed that (i) P-flavin is the product of luciferase, (ii) the physiological function of the lux operon is not to produce light but to produce FP(390) (luxF protein), including its prosthetic group, P-flavin, and (iii) FP(390) reactivates oxidatively inactivated cobalamin-dependent methionine synthase similar to flavodoxin but at relatively high ionic strength. It seems difficult to extend this idea to all luminous bacteria because the luxF gene is not present in the lux operon in Vibrio or Xenorhabdus. But we predicted that a luciferase fragment which binds P-flavin should function like FP(390) in these species. In this study, we isolated P-flavin binding protein from Vibrio fischeri ATCC 7744. The obtained protein was a modified luciferase as expected, in which the beta-subunit was intact but about 25 amino acid residues at the C-terminus of the alpha-subunit were deleted and the prosthetic group was the fully reduced P-flavin. These results strongly support that the physiological function of the lux operon is as described above even in luminous bacteria other than Photobacterium species. We propose that chromophore B reported by Tu and Hastings [Tu, S.-C. and Hastings, J.W. (1975) Biochemistry 14, 1975-1980] is the reduced P-flavin.  相似文献   

6.
Dispersed pump-dump-probe spectroscopy has the ability to characterize and identify the underlying ultrafast dynamical processes in complicated chemical and biological systems. This technique builds on traditional pump-probe techniques by exploring both ground- and excited-state dynamics and characterizing the connectivity between constituent transient states. We have used the dispersed pump-dump-probe technique to investigate the ground-state dynamics and competing excited-state processes in the excitation-induced ultrafast dynamics of thiomethyl p-coumaric acid, a model chromophore for the photoreceptor photoactive yellow protein. Our results demonstrate the parallel formation of two relaxation pathways (with multiple transient states) that jointly lead to two different types of photochemistry: cis-trans isomerization and detachment of a hydrated electron. The relative transition rates and quantum yields of both pathways have been determined. We find that the relaxation of the photoexcited chromophores involves multiple, transient ground-state intermediates and the chromophore in solution does not generate persistent photoisomerized products, but instead undergoes photoionization resulting in the generation of detached electrons and radicals. These results are of great value in interpreting the more complex dynamical changes in the optical properties of the photoactive yellow protein.  相似文献   

7.
Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosylmethionine. Another V. fischeri gene, ainS, directs the synthesis of N-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the Vibrio harveyi LuxM protein. The luxM gene is required for the synthesis of N-(3-hydroxybutyryl)-L-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of N-octanoylhomoserine lactone from S-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes.  相似文献   

8.
9.
Shuttle vectors that had previously been shown to replicate both in Escherichia coli and in strains of Anabaena spp. were used to transfer the lux genes from Vibrio harveyi and Vibrio fischeri into Anabaena spp. The level of expression of luciferase in the cyanobacteria (up to 7,000 quanta cell-1 s-1) makes these genes good candidates for use as promoter probes during the differentiation of certain cells in a filament into heterocysts.  相似文献   

10.
The blue fluorescent protein (BFPVV) gene bfpvv from Vibrio vulnificus CKM-1 was cloned and sequenced. The transformants exhibited blue fluorescence when irradiated by UV source. Nucleotide sequence analysis predicted an ORF of 717 bp encoding a 239-amino-acid polypeptide with a calculated molecular mass of 25.8 kDa. The nucleotide sequence of the bfpvv gene and its deduced amino acid sequence showed significant homology to those of the short-chain dehydrogenase/reductase (SDR) family proteins from various organisms. Some functionally important residues in SDR were strictly conserved in BFPVV, such as an active-site Tyr145, a catalytic site Lys149, and a common GlyXXXGlyXGly pattern in the N-terminal part of the molecule. By changing three amino acid residues, Tyr145, Lys149, and Gly9 to Phe, Ile, and Val, respectively, it was found that the G9V mutant did not generate blue fluorescence, while mutants Y145F and K149I have 126 and 68.5% fluorescence compared with the wild-type BFPVV.  相似文献   

11.
12.
Resonance Raman (RR) spectra of green fluorescent protein (GFP) model chromophores in solution have been simulated with the CASSCF/MM methodology. Although several reports on vibrational analysis of GFP model chromophores have been recently published, the RR spectra were simulated for the first time in explicit solution with the inclusion of the counterion, as these effects are crucial for unambiguously reproducing the vibrational band assignment in the anionic form of the GFP chromophore. This strategy allows for a one-to-one correspondence of the calculated vibrational modes to the observed RR bands, concerning both the location and intensity pattern. In addition, these simulations were complemented with total energy distribution calculations to aid in the unambiguous assignment of the measured spectra. The current study helps to clarify some of the previous RR bands assignments as well as producing some new assignment for the anionic form of GFP chromophore. The explicit solvent simulations and PCM-based calculations are compared to the measured spectra, and these results demonstrate that explicit solvent simulations provide better agreement with experiment, both in terms of vibrational frequencies and intensity distribution. Figure a Correlation of explicit hydration calculations (CASSCF/6-31G*/MM) for the HBI model chromophore and experimental RR data [21]; slope = 0.982, intercept = 27.210 and regression coefficient = 0.997. b Correlation of implicit PCM calculations (CASSCF/6-31G*) for the HBI model chromophore and experimental RR data [21], slope = 1.017, intercept = −48.838 and regression coefficient = 0.984  相似文献   

13.
Dissimilatory nitrite reductase was isolated from anaerobically nitrate-grown Vibrio fischeri cells and purified to electrophoretic homogeneity. The enzyme catalyzes the six-electron reduction of nitrite to ammonia. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under either nonreducing or reducing conditions, the purified nitrite reductase migrated as a single protein band of Mr 57,000. Gel filtration chromatography revealed a native molecular weight of 58,000, indicating the enzyme as isolated to be present in the monomeric form. Purified nitrite reductase exhibited typical c-type cytochrome absorption spectra with the reduced alpha-band at 552.5 nm. Heme content analysis using the purified preparation indicated the enzyme to contain 5.5 heme c groups per molecule. Iron analysis showed the presence of 5.62 g iron atoms per mole of enzyme and no nonheme irons were detected. These results clearly indicate that, similar to the dissimilatory nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes, and Escherichia coli, the V. fischeri nitrite reductase is a hexaheme c-type cytochrome. Amino acid composition of V. fischeri also revealed close similarities to those of the other three hexaheme nitrite reductases previously studied. Based on this information, it is concluded that the four ammonia-forming, dissimilatory nitrite reductases isolated to date represent a homologous group of proteins with the distinct property of being hexaheme c-type cytochromes.  相似文献   

14.
The fluorescence decays of several analogues of the photoactive yellow protein (PYP) chromophore in aqueous solution have been measured by femtosecond fluorescence up-conversion and the corresponding time-resolved fluorescence spectra have been reconstructed. The native chromophore of PYP is a thioester derivative of p-coumaric acid in its trans deprotonated form. Fluorescence kinetics are reported for a thioester phenyl analogue and for two analogues where the thioester group has been changed to amide and carboxylate groups. The kinetics are compared to those we previously reported for the analogues bearing ketone and ester groups. The fluorescence decays of the full series are found to lie in the 1-10 ps range depending on the electron-acceptor character of the substituent, in good agreement with the excited-state relaxation kinetics extracted from transient absorption measurements. Steady-state photolysis is also examined and found to depend strongly on the nature of the substituent. While it has been shown that the ultrafast light-induced response of the chromophore in PYP is controlled by the properties of the protein nanospace, the present results demonstrate that, in solution, the relaxation dynamics and pathway of the chromophore is controlled by its electron donor-acceptor structure: structures of stronger electron donor-acceptor character lead to faster decays and less photoisomerisation.  相似文献   

15.
16.
To investigate the roles of amino acid residues around the chromophore in photoactive yellow protein (PYP), new mutants, Y42A, E46A, and T50A were prepared. Their spectroscopic properties were compared with those of wild-type, Y42F, E46Q, T50V, R52Q, and E46Q/T50V, which were previously prepared and specified. The absorption maxima of Y42A, E46A, and T50A were observed at 438, 469, and 454 nm, respectively. The results of pH titration for the chromophore demonstrated that the chromophore of PYP mutant, like the wild-type, was protonated and bleached under acidic conditions. The red-shifts of the absorption maxima in mutants tended toward a pK(a) increase. Mutation at Glu46 induced remarkable shifts in the absorption maxima and pK(a). The extinction coefficients were increased in proportion to the absorption maxima, whereas the oscillator strengths were constant. PYP mutants that conserved Tyr42 were in the pH-dependent equilibrium between two states (yellow and colorless forms). However, Y42A and Y42F were in the pH-independent equilibrium between additional intermediate state(s) at around neutral pH, in which yellow form was dominant in Y42F whereas the other was dominant in Y42A. These findings suggest that Tyr42 acts as the hinge of the protein, and the bulk as well as the hydroxyl group of Tyr42 controls the protein conformation. In all mutants, absorbance at 450 nm was decreased upon flash irradiation and afterwards recovered on a millisecond time scale. However, absorbance at 340--370 nm was increased vice versa, indicating that the long-lived near-UV intermediates are formed from mutants, as in the case of wild-type. The lifetime changes with mutation suggest the regulation of proton movement through a hydrogen-bonding network.  相似文献   

17.
Wavelength- and time-resolved fluorescence experiments have been performed on the photoactive yellow protein, the E46Q mutant, the hybrids of these proteins containing a nonisomerizing “locked” chromophore, and the native and locked chromophores in aqueous solution. The ultrafast dynamics of these six systems is compared and spectral signatures of isomerization and solvation are discussed. We find that the ultrafast red-shifting of fluorescence is associated mostly with solvation dynamics, whereas isomerization manifests itself as quenching of fluorescence. The observed multiexponential quenching of the protein samples differs from the single-exponential lifetimes of the chromophores in solution. The locked chromophore in the protein environment decays faster than in solution. This is due to additional channels of excited-state energy dissipation via the covalent and hydrogen bonds with the protein environment. The observed large dispersion of quenching timescales observed in the protein samples that contain the native pigment favors both an inhomogeneous model and an excited-state barrier for isomerization.  相似文献   

18.
Wan S  Liu S  Zhao G  Chen M  Han K  Sun M 《Biophysical chemistry》2007,129(2-3):218-223
Photoabsorption properties of green and red fluorescent protein chromophore anions in vacuo were investigated theoretically, based on the experimental results in gas phase [Phys. Rev. Lett. 2001, 87, 228102; Phys. Rev. Lett. 2003, 90, 118103]. Their calculated transition energies in absorption with TD-DFT and ZINDO methods are directly compared to the experimental reports in gas phase, and the calculations with ZINDO method can correctly reproduce the absorption spectra. The orientation and strength of their transition dipole moments were revealed with transition density. We also showed the orientation and result of their intramolecular charge transfer with transition difference density. The calculated results show that with the increase of the extended conjugated system, the orientation of transition dipole moments and the orientation of charge transfer can be reversed. They are the linear responds with the external electric fields. These theoretical results reveal the insight understanding of the photoinduced dynamics of green and red fluorescent protein chromophore anions and cations in vacuo.  相似文献   

19.
Expression of Vibrio fischeri luminescence genes requires an inducer, termed autoinducer, and a positive regulatory element, the luxR gene product. A plasmid containing a tac promoter-controlled luxR was mutagenized in vitro with hydroxylamine, and luxR mutant plasmids were identified by their inability to complement a luxR deletion mutation in trans. Sixteen luxR mutant plasmids were obtained, ten of which encoded full-length but inactive luxR gene products as demonstrated by a Western immunoblot analysis. The effects of 1 of the 10 mutations could be overcome by the addition of autoinducer at a high concentration. The mutations in each of the 10 mutant plasmids that directed the synthesis of an inactive LuxR protein were identified by DNA sequencing. Of the 10 proteins encoded by the mutant luxR plasmids, 9 differed from the normally active LuxR in only a single amino acid residue. The amino acid residue substitutions in the proteins encoded by the nine mutant luxR genes clustered in two regions. One region around the middle of the polypeptide encoded by luxR was hypothesized to represent an autoinducer-binding domain, and the other region towards the carboxy terminus of the gene product was hypothesized to constitute a lux operator DNA-binding domain or a lux operator DNA recognition domain.  相似文献   

20.
We present results of theoretical studies of the photoabsorption band corresponding to the vertical electronic transition S0–S1 between first two singlet states of the model chromophore from the green fluorescent protein (GFP) in its neutral form. Predictions of quantum chemical approaches including ab initio and semi-empirical approximations are compared for the model systems which mimic the GFP chromophore in different environments. We provide evidences that the protein matrix in GFP accounts for a fairly large shift of about 40 nm in the S0–S1 absorption band as compared to the gas phase.  相似文献   

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