Lumazine Protein Research Articles

Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators.

The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (tau = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of tau had virtually no effect on the rotational correlation time of the lumazine protein (phi = 9.4 ns, 19 degrees C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 degrees C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (tau = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).

IDENTIFICATION OF A LUMAZINE PROTEIN FROM PHOTOBACTERIUM LEIOGNATHI BY COHERENT ANTI‐STOKES RAMAN SPECTROSCOPY

AbstractThe vibrational fingerprint in the frequency region 1700‐1100 cm‐1 has been obtained for a fluorescent protein purified from the bioluminescent bacterium Photobacterium leiognathi. using the technique of resonance coherent anti‐Stokes Raman scattering. The frequencies of the 4 prominent modes are the same as for lumazine protein purified from Photobacterium phosphoreum, and for an authentic sample of 6,7‐dimethyl‐8‐ribityllumazine. The fluorophore in the new protein, therefore, must have the same chemical structure. The absence of frequency shifts between the free and bound ligand, in contrast to observations of fiavoproteins, implies that the lumazine ring undergoes no changes in hydrogen bonding between the free and protein‐bound ligand in its ground electronic state. However, changes in relative intensities of two modes are seen, arising from differences in interactions in the excited state, and this is also reflected by differences in fluorescence properties.

SENSITIZATION BY LUMAZINE PROTEINS OF THE BIOLUMINESCENCE EMISSION FROM THE REACTION OF BACTERIAL LUCIFERASES

Abstract— Lumazine protein from Photobacterium phosphoreurn blue shifts the in vitro bioluminescence spectra in the reactions using each of the 4 main types of bacterial luciferases: P. phosphoreum, P. leiognathi, Vibrio harveyi and V. fischeri. For the reaction initiated with FMNH2 and tetradecanal at 2°C, this “sensitizing” property of lumazine protein differs quantitatively between the luciferases. An interaction constant characterizing each type of luciferase may be derived from a reciprocal plot of the spectral shift against the lurnazine protein concentration. The weakest interaction constant is in the V. fischeri reaction, 180 μM. For the V. harveyi reaction the interaction is in the range 6–9 μM, and for both Photobacterium reactions it is 2–3 μM. A concentration of only 0.6 μM of lumazine protein is sufficient to cause an observable change in the Photobacterium bioluminescence spectra. For the V. harveyi case the interaction constant is near to the equilibrium Kd for the luciferase‐lumazine protein complex, observed directly by Visser and Lee. Both constants are decreased markedly by increase in phosphate concentration so that it is concluded that, with V. harveyi luciferase, sensitization occurs within this protein‐protein complex. For P. phosphoreum luciferase, however, the equilibrium complex is too weak to correspond to the sensitizing interaction and it is concluded that the rate‐limiting process is a protein‐protein bimolecular collision. As judged from their molecular weight around 20000, spectral properties, and ability to blue shift the bioluminescence spectra, lumazine proteins are identified in a second strain of P. phosphoreum and in P. leiognathi.

Protein-ligand interactions in lumazine protein and in Desulfovibrio flavodoxins from resonance coherent anti-Stokes Raman spectra.

The resonance coherent anti-Stokes Raman technique was used to obtain vibrational spectra of flavin in flavodoxins from Desulfovibrio gigas and Desulfovibrio vulgaris and of the simpler 6,7-dimethyl-8-ribityllumazine chromophore in the blue fluorescence lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. In the region examined, 1100-1700 cm-1, the Raman spectrum of the lumazine is less crowded than that of the flavin and this facilitates assignment of observed frequencies to particular vibrational modes. Certain modes are not affected by chromophore binding to the protein, but others are changed in frequency or intensity in a way that can be rationalized by expected interactions of the chromophore with the amino acid residues of the binding site. For example, a tentative assignment of change in hydrogen bonding at N(5) is suggested as the cause of the frequency shift for the chromophore in both flavodoxins (free-bound, 1582-1572 cm-1) and for C(4)=O in glucose oxidase (1359-1364 cm-1) and lumazine protein (1359-1362 cm-1). Shifts of the C(2)-N(3) mode in D2O may arise from hydrogen-bonding changes at C(2)=O in lumazine protein (1284-1291 cm-1), flavodoxin (1300-1280 cm-1), and glucose oxidase (1297-1287 cm-1). Bonding at N(3)-H may be the origin of changes in the frequency or intensity of the amide III mode in riboflavin binding protein and glucose oxidase. A stacking interaction is suggested for the change in a pyrimidine ring mode in FAD (1508 cm-1) since this mode is found at 1504 cm-1 in 30% Me2SO/H2O, where the adenine and pyrimidine are unstacked. The results clearly indicate different interactions in the binding sites of those proteins studied to date.

Lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. Purification and characterization.

Lumazine protein, a novel protein containing 6,7-dimethyl-8-ribityllumazine as a bound prosthetic group, is one of the several major proteins produced by the bioluminescent bacteria, Photobacterium phosphoreum. purification to complete homogeneity from cell extracts is achieved in six steps. Lumazine protein is a near spherical, monomeric protein of average molecular weight 20,000; in amino acid composition it is acidic with two isoelectric isomers, pI 4.9 and 5.0, and is hydrophilic (974 cal/residue) with single methionine and tryptophan residues and two accessible cysteines. It contains no carbohydrate. Reaction of the cysteines with dithionitrobenzoic acid results in quenching of the bound lumazine fluorescence but is otherwise reversible. Estimates of protein by dry weight results in a mole ratio of one bound lumazine group per protein.

Lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. A fluorescence study of the protein-ligand equilibrium.

The changes of fluorescence spectral distribution, polarization, and lifetime of the lumazine protein from Photobacterium phosphoreum can be interpreted in terms of an equilibrium between the protein and its dissociated prosthetic group 6,7-dimethyl-8-(1'-D-ribityl)lumazine. The equilibrium is rapidly attained, 1:1, and Kd is 5 x 10(-8) M (4 degrees C, pH 7, 67 mM phosphate). A change in solution conditions like an increase in temperature or dilution or a decrease in pH or ionic strength favors dissociation of the ligand from the protein. The dissociation was confirmed by separating the free ligand by ultrafiltration, and the apoprotein was reconstituted with the authentic lumazine derivative. A van't Hoff analysis of the dissociation constant allows evaluation of the thermodynamic parameters: delta H degrees = 28 kcal/mol and delta S degrees = 67 eu. By analogy to published results on the binding of FMN to apoflavodoxin, the contribution of hydrophobic interaction and of hydrogen bonding to the binding enthalpy can be estimated. The decay of the emission anisotropy of lumazine protein following a 0.5-ns laser pulse excitation can be fitted with a single correlation time characteristic of a 30 000-dalton spherical protein.