Spectral properties and function of two lumazine proteins from Photobacterium

The spectral properties are compared for two 6,7-dimethyl-8-ribityllumazine proteins from marine bioluminescent bacteria, one from a psychrophile, Photobacterium phosphoreum, and the other from a thermophile, Photobacterium leiognathi. The visible spectral properties, which are the ones by which the protein performs its biological function of bioluminescence emission, are almost the same for the two proteins: at 2 degrees C and 50 mM Pi, pH 7, fluorescence quantum yield phi F = 0.59 and 0.54, respectively; fluorescence lifetime tau = 14.4 and 14.8 ns, respectively; fluorescence maxima, both 475 nm; absorption maximum, 417 and 420 nm, respectively; circular dichroism minima at around 420 nm, both -41 X 10(3) deg cm2 dmol-1. The ligand binding sites therefore must provide very similar environments, and arguments are presented that the bound ligand is relatively exposed to solvent. The dissociation equilibrium was studied by steady-state fluorescence polarization. The thermophilic protein binds the ligand with Kd (20 degrees C) = 0.016 microM, 10 times more tightly than the other protein [Kd (20 degrees C) = 0.16 microM]. The origin of the binding difference probably resides in differences in secondary structure. The tryptophan fluorescence spectra of the two proteins are different, but more significant is an observation of the decay of the tryptophan emission anisotropy. For the psychrophilic lumazine protein this anisotropy decays to zero in 1 ns, implying that its single tryptophan residue lies in a very "floppy" region of the protein. For the other protein, the anisotropy exhibits both a fast component and a slow one corresponding to rotation of the protein as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic investigations of the single tryptophan residue and of riboflavin and 7-oxolumazine bound to lumazine apoprotein from Photobacterium leiognathi

Spectroscopic techniques have been applied to investigate the conformation, local structure, and dynamic properties of the apoprotein of the lumazine protein from Photobacterium leiognathi and the holoprotein reconstituted with either the natural ligand 6,7-dimethyl-8-ribityllumazine or the closely related analogues riboflavin and 6-methyl-7-oxo-8-ribityllumazine (7-oxolumazine). The analogues are bound similarly to the natural prosthetic group. They exhibit similar shifts on binding in their absorption and fluorescence spectra, single-exponential fluorescence decays, and no independent motion from the protein as evident from a long-lived anisotropy decay (single-exponential phi = 10 ns, 20 degrees C) and high initial anisotropy. Steady-state anisotropy measurements result in similar KD's (40 nM, 20 degrees C, 50 mM inorganic phosphate) for all ligands. Circular dichroism in the far-UV region (190-250 nm) indicates no change in secondary structure on binding to the apoprotein. In the spectral region of 250-310 nm relatively large changes occur, indicating changes in the environment of the tyrosine and tryptophan residues. The single tryptophan residue shows a three-exponential decay of its fluorescence in both the apoprotein and the holoprotein. Radiationless energy transfer also occurs from the tryptophan to the bound ligand, especially evident with 7-oxolumazine. We have designed a new method for evaluation of the rate constant of energy transfer by measuring the (picosecond) rise time of the acceptor fluorescence. The anisotropy decay of the tryptophan residue shows two correlation times, a short one (phi approximately equal to 0.4 ns) representing rapid but restriced oscillation of this residue and a longer one (phi 2 = 5-7 ns, 20 degrees C) representing the motion of a larger segment of the protein.     

Fluorescence study of the ligand stereospecificity for binding to lumazine protein.

6,7-Dimethyllumazine derivatives, substituted at the 8-position with aldityls or monohydroxyalkyl groups, have been examined for their binding ability to lumazine apo-protein from two strains of Photobacterium phosphoreum using fluorescence dynamics techniques. On the protein the lumazine has a nearly monoexponential decay of fluorescence with lifetime 13.8 ns (20 degrees C). In free solution the lifetime is 9.6 ns. The concentration of free and bound lumazine in an equilibrium mixture can be recovered readily by analysis of the fluorescence decay. Only the aldityl derivatives D-xylityl and 3'-deoxy-D-ribityl, having stereoconfigurations at the 2' and 4' positions identical to the natural ligand, 8-(1'-D-ribityl), show comparable dissociation constants (0.3 microM, 20 degrees C, pH 7.0). D-Erythrityl and L-arabityl have dissociation constants of 1-2 microM. All other ligands show no interaction at all or have dissociation constants in the range 6-80 microM, which can still be determined semi-quantitatively using the fluorescence decay technique. In the case of these very weakly bound ligands, unambiguous detection of bound ligand can be shown by a long correlation time (23 ns, 2 degrees C) for the fluorescence anisotropy decay. Examination of the bound D-xylityl compound's fluorescence anisotropy decay at high time resolution (< 100 ps) shows rigid association, i.e. no mobility independent of the macromolecule. All bound ligands appear to be similarly positioned in the binding site. The influence of the stereoconfiguration at the 8-position found for lumazine protein parallels that previously observed for the enzyme riboflavin synthase, where the lumazines are substrates or inhibitors. This is consistent with the finding of significant sequence similarity between these proteins. The binding rigidity may have implications for the mechanism of the enzyme.     

Electronic excitation transfer in the complex of lumazine protein with bacterial bioluminescence intermediates

Fluorescence dynamics measurements have been made on the bioluminescence reaction intermediates using Photobacterium leiognathi, Vibrio fischeri, and Vibrio harveyi luciferases, both alone and in mixtures with Photobacterium phosphoreum lumazine protein. Each luciferase produces a "fluorescent transient" intermediate on reaction with the bioluminescence substrates, FMNH2, tetradecanal, and O2, and all have a fluorescence quantum yield about 0.3, with a predominant lifetime around 10 ns. The P. leiognathi luciferase fluorescent transient has a rotational correlation time of 79 ns at 2 degrees C, as expected for the rotational diffusion of a 77-kDa macromolecule. In the presence of lumazine protein however a faster correlation time of about 3 ns predominates. This rapid channel of anisotropy loss is attributed to energy transfer from the flavin intermediate bound on the luciferase to the lumazine ligand, reflects the presence of protein-protein complexation, and is greatest in the case of P. leiognathi, but not at all for V. fischeri. This fact is consistent with the strong influence of lumazine protein on the bioluminescence reaction of P. leiognathi, and not at all with V. fischeri. The rate of energy transfer is of order 10(9) s-1, much greater than the 10(8) s-1 fluorescence rate of the donor. Thus the bioluminescence excitation of lumazine protein could occur by a similar photophysical mechanism of interprotein energy transfer from a chemically excited fluorescent transient donor to the lumazine acceptor.     

Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence

The mechanism of the shifting of the bioluminescence spectrum from the reaction of bacterial luciferase by lumazine protein is investigated by methods of fluorescence dynamics. A metastable intermediate is produced on reaction of Vibrio harveyi luciferase with FMNH2 and O2. It has an absorption maximum at 374 nm and a rotational correlation time (phi) derived from the decay of its fluorescence (maximum 500 nm) anisotropy of 90 ns (2 degrees C). Lumazine protein from Photobacterium phosphoreum has an absorption maximum at 417 nm and a fluorescence maximum at 475 nm. Lumazine protein forms a protein-protein complex with luciferase, and the complex has a phi of approximately 100 ns. A mixture of lumazine protein and the intermediate would be expected to have an average correlation time (phi av) around 100 ns, but instead, the result is anomalous. The phi av is much lower and is also wavelength dependent. For excitation at 375 nm, which is mainly absorbed in the flavin chromophore of the intermediate, phi av = 25 ns, but at 415 nm, mainly absorbed by the lumazine derivative ligand of lumazine protein, phi av approximately 50 ns. It is proposed that protein-protein complexation occurs between lumazine protein and the luciferase intermediate and that in this complex energy transfer from the flavin to the lumazine is the predominant channel of anisotropy loss. A distance of 20 A between the donor and acceptor is calculated. In the bioluminescence reaction of intermediate with tetradecanal, a fluorescent transient species is produced which is the bioluminescence emitter.(ABSTRACT TRUNCATED AT 250 WORDS)     

13C and 15N NMR studies on the interaction between 6,7-dimethyl-8-ribityllumazine and lumazine protein

The interaction between the prosthetic group 6,7-dimethyl-8-(1'-D-ribityl)lumazine and the lumazine apoproteins from two marine bioluminescent bacteria, one from a relatively thermophilic species, Photobacterium leiognathi, and the other from a psychrophilic species, Photobacterium phosphoreum, was studied by 13C and 15N NMR using various selectively enriched derivatives. It is shown that the electron distribution in the protein-bound 6,7-dimethyl-8-ribityllumazine differs from that of free 6,7-dimethyl-8-ribityllumazine in buffer. The 13C and 15N chemical shifts indicate that the protein-bound 6,7-dimethyl-8-ribityllumazine is embedded in a polar environment and that the ring system is strongly polarized. It is concluded that the two carbonyl groups play an important role in the polarization of the molecule. The N(3)-H group is not accessible to bulk solvent. The N(8) atom is sp2 hybridized and has delta+ character. Nuclear Overhauser effect studies indicate that the 6,7-dimethyl-8-ribityllumazine ring is rigidly bound with no internal mobility. The NMR results indicate that the interaction between the ring system and the two apoproteins is almost the same.     

Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum

The properties of lumazine proteins purified from the marine bioluminescent bacteria Photobacterium phosphoreum, a psychrophile, and Photobacterium leiognathi, a relatively thermophilic species, are compared. An accurate 1:1 stoichiometry of binding of the ligand 6,7-dimethyl-8-ribityllumazine to each lumazine protein is established by back-titration of the apoprotein with the authentic ligand, using both fluorescence and absorption measurements. Neither protein contains metal cofactors, organic phosphorus, or carbohydrate. Both proteins are anionic and hydrophilic. They each contain a single Trp residue and have blocked amino terminals but otherwise differ in amino acid composition and other properties (P. phosphoreum and P. leiognathi, respectively): Met (internal), 1, 2; Cys, 2, 1; Arg, 4, 7; pI, 4.78 and 4.83, 4.38 and 4.45; Mr, 19 750, 21 300. In the P. phosphoreum protein both Cys residues are accessible, but in the P. leiognathi protein the single Cys is "buried". Modification of this buried Cys and at least one Cys in the P. phosphoreum protein prevents binding of the ligand. The UV and visible absorption spectra of both lumazine proteins denatured in 6 M guanidine hydrochloride can be accurately modeled by using the number of equivalents of the lumazine derivative and blocked aromatic amino acid model compounds determined by chemical and spectrophotometric analyses for Trp, Tyr, and Phe.     

Time-resolved fluorescence spectroscopy of lumazine protein from Photobacterium phosphoreum using synchrotron radiation

Time-resolved fluorescence on lumazine protein from Photobacterium phosphoreum was performed with synchrotron radiation as a source of continuously tunable excitation. The experiments yielded structural and dynamic details from which two aspects became apparent. From fluorescence anisotropy decay monitoring of lumazine fluorescence with different excitation wavelengths, the average correlation times were shown to change, which must indicate the presence of anisotropic motion of the protein. A similar study with 7-oxolumazine as the fluorescent ligand led to comparable results. The other remarkable observation dealt with the buildup of acceptor fluorescence, also observed with 7-oxolumazine although much less pronounced, which is caused by the finite energy transfer process between the single donor tryptophan and the energy accepting lumazine derivatives. Global analytical approaches in data analysis were used to yield realistic correlation times and reciprocal transfer rate constants. It was found that the tryptophan residue has a large motional freedom as also reported previously for this protein and for the related protein from P. leiognathi (Lee et al. 1985; Kulinski et al. 1987). The average distance between the tryptophan residue and the ligand donor-acceptor couple has been determined to be 2.7 nm for the same donor and two different acceptors.     

Dynamic fluorescence study of the interaction of lumazine protein with bacterial luciferases

The equilibrium association of lumazine protein from Photobacterium phosphoreum with luciferases from either P. phosphoreum or an aldehyde-requiring dark mutant of Vibrio harveyi is measured from changes of the rotational correlation time which is derived from the decay of the lumazine ligand's fluorescence anisotropy. The rotational correlation time of lumazine protein is 23 ns (2 degrees C, 0.25 M Pi) and is increased on addition of luciferase due to the formation of a higher molecular weight complex. The V. harveyi luciferase exhibits full competence for the association and a 1:1 stoichiometry with a Kd in the range 40-90 microM. At lower ionic strength (0.05 M Pi), the Kd increases but is reduced again by the addition of dodecanol or dimyristoyllecithin. In contrast, tetradecanal, a substrate for the bioluminescence reaction, exerts no influence on the association. The equilibration rate is found to be too slow and for both luciferases the Kd values are too high for the interaction of the native proteins to account quantitatively for the spectral shifting of the bioluminescence by lumazine protein.     

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.     

One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins

We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at approximately 440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore.     

Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.     

Dynamics of supercoiled and relaxed pTZ18U plasmids probed with a long-lifetime metal-ligand complex

[Ru(bpy)2(dppz)](2+) (bpy = 2,2'-bipyridine, dppz = dipyrido- [3,2-a:2',3'-c]phenazine) (RuBD), a long-lifetime metalligand complex, displays favorable photophysical properties. These include long lifetime, polarized emission, but no significant fluorescence from the complex that is not bound to DNA. To show the usefulness of this luminophore (RuBD) for probing the bending and torsional dynamics of nucleic acids, its intensity and anisotropy decays when intercalated into supercoiled and relaxed pTZ18U plasmids were examined using frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The mean lifetimes for the supercoiled plasmids (< tau > = 148 ns) were somewhat shorter than those for the relaxed plasmids (< tau > = 160 ns). This suggests that the relaxed plasmids were shielded more efficiently from water. The anisotropy decay data also showed somewhat shorter slow rotational correlation times for supercoiled plasmids (288 ns) than for the relaxed plasmids (355 ns). The presence of two rotational correlation times suggests that RuBD reveals both the bending and torsional motions of the plasmids. These results indicate that RuBD can be useful for studying both the bending and torsional dynamics of nucleic acids.     

Lumazine proteins from photobacteria: localization of the single ligand binding site to the N-terminal domain

Lumazine protein is believed to serve as an optical transponder in bioluminescence emission by certain marine bacteria. Sequence arguments suggest that the protein comprises two similarly folded riboflavin synthase-type domains, but earlier work also suggested that only one domain binds 6,7-dimethyl-8-ribityllumazine (DMRL). We show that the replacement of serine-48 or threonine-50 in the N-terminal domain of lumazine protein of Photobacterium leiognathi modulates the absorbance and fluorescence properties of bound DMRL or riboflavin. Moreover, the replacement of these amino acids is accompanied by reduced ligand affinity. Replacement of serine-48 by tryptophan shifts the (13)C NMR signal of the 6-methyl group in bound DMRL upfield by 2.9 ppm as compared to the wild-type protein complex. Replacement of threonine-50 causes a downfield shift of approximately 20 ppm for the (15)N NMR signal of N-5, as well as an upfield shift of 3 ppm for the (13)C NMR signal of C-7 in bound DMRL, respectively. The replacement of the topologically equivalent serine-144 and proline-146 in the C-terminal domain had no significant impact on optical properties, chemical shifts and apparent binding constants of bound DMRL. These data show that the N-terminal domain is the unique site for ligand binding in lumazine protein.     

Evolution of vitamin B2 biosynthesis: 6,7-dimethyl-8-ribityllumazine synthases of Brucella

The penultimate step in the biosynthesis of riboflavin (vitamin B2) involves the condensation of 3,4-dihydroxy-2-butanone 4-phosphate with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase). Pathogenic Brucella species adapted to an intracellular lifestyle have two genes involved in riboflavin synthesis, ribH1 and ribH2, which are located on different chromosomes. The ribH2 gene was shown previously to specify a lumazine synthase (type II lumazine synthase) with an unusual decameric structure and a very high Km for 3,4-dihydroxy-2-butanone 4-phosphate. Moreover, the protein was found to be an immunodominant Brucella antigen and was able to generate strong humoral as well as cellular immunity against Brucella abortus in mice. We have now cloned and expressed the ribH1 gene, which is located inside a small riboflavin operon, together with two other putative riboflavin biosynthesis genes and the nusB gene, specifying an antitermination factor. The RibH1 protein (type I lumazine synthase) is a homopentamer catalyzing the formation of 6,7-dimethyl-8-ribityllumazine at a rate of 18 nmol mg(-1) min(-1). Sequence comparison of lumazine synthases from archaea, bacteria, plants, and fungi suggests a family of proteins comprising archaeal lumazine and riboflavin synthases, type I lumazine synthases, and the eubacterial type II lumazine synthases.     

Structure of lumazine protein, an optical transponder of luminescent bacteria

The intensely fluorescent lumazine protein is believed to be involved in the bioluminescence of certain marine bacteria. The sequence of the catalytically inactive protein resembles that of the enzyme riboflavin synthase. Its non-covalently bound fluorophore, 6,7-dimethyl-8-ribityllumazine, is the substrate of this enzyme and also the committed precursor of vitamin B₂. An extensive crystallization screen was performed using numerous single-site mutants of the lumazine protein from Photobacterium leiognathi in complex with its fluorophore and with riboflavin, respectively. Only the L49N mutant in complex with riboflavin yielded suitable crystals, allowing X-ray structure determination to a resolution of 2.5 Å. The monomeric protein folds into two closely similar domains that are structurally related by pseudo-C₂ symmetry, whereby the entire domain topology resembles that of riboflavin synthase. Riboflavin is bound to a shallow cavity in the N-terminal domain of lumazine protein, whereas the C-terminal domain lacks a ligand.     

Flexibility of the molecular forms of acetylcholinesterase measured with steady-state and time-correlated fluorescence polarization spectroscopy

Steady-state and time-correlated fluorescence polarizations have been examined for selected complexes and covalent conjugates of the 11S and (17 + 13)S forms of Torpedo acetylcholinesterase. The 11S form exists as a tetramer of apparently identical subunits, whereas the (17 + 13)S forms contain two or three sets of tetramers disulfide-linked to an elongated collagen-like tail unit. Pyrenebutyl methylphosphonofluoridate and (dansylsulfonamido)pentyl methylphosphonofluoridate were conjugated at the active center serine whereas propidium was employed as a fluorescent ligand for the spatially removed peripheral anionic site. Steady-state polarization of the pyrenebutyl conjugates indicates rotational correlation times of approximately 400 ns for the 11S species and greater than 1100 ns for the (17 + 13)S species. Hence, the tail unit severely restricts rotational motion of the catalytic subunits. Time-correlated fluorescence polarization analysis of the 11S species indicates multiple rotational correlation times. Anisotropy decay of the propidium complex (tau = 6 ns) occurs in exponential manner with a rotational correlation time of approximately 150 ns, while covalent adducts at the active center exhibit rotational correlation times greater than or equal to 300 ns. Anisotropy decay of the (dansylsulfonamido)pentyl conjugate (tau = 16 ns) appears exponential with a correlation time of approximately 320 ns, whereas decay of the pyrenebutyl conjugate (tau = 100 ns) is described by two correlation times, phi S = 18 ns and phi L = 320 ns, of small (15%) and large (85%) amplitudes, respectively. Two limiting models have been considered to explain the results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of 4-(1'-D-ribitylamino)-5-amino-2,6-dihydroxypyrimidine from a riboflavin-adenine-deficient mutant of Bacillus subtilis.

Studies were carried out to determine possible intermediates involved in the biosynthetic pathway of riboflavin, using resting cells of a riboflavin-adenine-deficient mutant, Bacillus subtilis AJ1988. The cells excreted 6,7-dimethyl-8-ribityllumazine, the end product in the biosynthetic pathway, into the incubation medium in large amounts. The addition of glyoxal caused a large accumulation of a green fluorescent compound; an inverse relation was observed between the formation of the lumazine and the concentration of glyoxal. Furthermore, added [2-14C]guanine effectively incorporated into the lumazine and the fluorescent compound in the same specific activity during incubation. The fluorescent compound was isolated, purified, and identified by paper chromatographic, fluorometric, and spectrophotometric analyses. It was proved to be 8-(1'-D-ribityl)lumazine, which appeared to have been formed by a reaction between glyoxal and a possible intermediate in the cells. Accordingly, 4-(1'-D-ribitylamino)-5-amino-2,6-dihydroxypyrimidine was concluded to be an immediate precursor of 6,7-dimethyl-8-ribityllumazine.     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

Fluorescence anisotropy studies of molecularly imprinted polymers.

A molecularly imprinted polymer (MIP) is a biomimetic material that can be used as a biochemical sensing element. We studied the steady-state and time-resolved fluorescence and fluorescence anisotropy of anthracene-imprinted polyurethane. We compared MIPs with imprinted analytes present, MIPs with the imprinted analytes extracted, MIPs with rebound analytes, non-imprinted control polymers (non-MIPs) and non-MIPs bound with analytes to understand MIP's binding behaviour. MIPs and non-MIPs had similar steady-state fluorescence anisotropy in the range 0.11-0.24. Anthracene rebound in MIPs and non-MIPs had a fluorescence lifetime of tau = 0.64 ns and a rotational correlation time of phi(F) = 1.2-1.5 ns, both of which were shorter than that of MIPs with imprinted analytes present (tau = 2.03 ns and phi(F) = 2.7 ns). The steady-state anisotropy of polymer solutions increased exponentially with polymerization time and might be used to characterize the polymerization extent in situ.