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)     

Recovery of components of fluorescence spectra of mixtures by intensity- and anisotropy decay-associated analysis: the bacterial luciferase intermediates

Reaction of FMNH2 and O2 with bacterial luciferase followed by blue light irradiation results in a product previously claimed to have the same fluorescence spectral distribution as the bioluminescence. Preparations of this "high fluorescence" intermediate, however, contain two fluorescent components, one from the intermediate and the other its breakdown product, FMN. Since the intermediate has a fluorescence lifetime of around 10 ns and a rotational correlation time in the range of 100 ns, compared to 5.0 and 0.15 ns, respectively, for the FMN, the two components can be successfully resolved from the total fluorescence by an anisotropy decay- and fluorescence decay-associated analysis employing simultaneous global computational methods. The fluorescence spectra of the intermediates from two types of luciferase were analyzed in this way; one luciferase was from Vibrio harveyi and the other was from an unusual type of V. fischeri that had an in vivo bioluminescence maximum at 505 nm, a wavelength almost 20 nm longer than that of the V. harveyi bioluminescence. For V. harveyi the true fluorescence of the intermediate is distinct from the bioluminescence, being found at a wavelength about 10 nm longer. For the type of V. fischeri examined, any difference in the two spectra is less certain. A control experiment with the dye 8-amino-1- naphthalenesulfonate bound to BSA and mixed with FMN recovered the original spectrum of the bound dye accurately.     

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.     

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).     

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.     

Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria is an obligate dimer and does not form a stable complex with the Ca(2+)-discharged photoprotein clytin

Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Fo?rster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.     

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.     

Effect of quinone on the fluorescence decay dynamics of endogenous flavin bound to bacterial luciferase

The interaction of quinone with luciferase from Photobacterium leiognathi was studied based on the fluorescence decay measurements of the endogenous flavin bound to the enzyme. Homologous 1,4-quinones, 1,4-benzoquinone, methyl-1,4-benzoquinone, 2-methyl-5-isopropyl-1,4-benzoquine and 1,4-naphthoquinone, were investigated. In the absence of quinone, the fluorescence intensity and anisotropy decays of the endogenous flavin exhibited two intensity decay lifetimes (~ 1 and 5 ns) and two anisotropy decay lifetimes (~ 0.2 and 20 ns), suggesting a heterogeneous quenching and a rotational mobility microenvironment of the active site of the luciferase, respectively. In the presence of quinone, the intensity decay heterogeneity was largely maintained, whereas the fraction of the short anisotropy decay component and the averaged rotational rate of FMN increased with the increasing hydrophobicity of the quinone. We hypothesize that the hydrophobicity of the quinone plays a role in the non-specific inhibition mechanism of xenobiotic molecules in the bacterial bioluminescence system via altering the rotational mobility of the endogenous flavin in the luciferase.     

Nanosecond spectroscopy of retinol.

Nanosecond fluorescence spectroscopy was used to study the unique binding site of the retinol-binding protein (RBP) from human serum. At pH 7.4, the binding of retinol to RBP caused the following spectroscopic changes in the ligand: (a) an enhancement of the fluorescence decay time (gamma = 8 ns); and (b) an increase in the emission anisotropy (A = 0.29). Retinol in hexane has a fluorescent decay time of 4.2 ns and a low emission anisotropy (A = 0.02). The increase in the fluorescence decay time of bound retinol is not due to dielectric relaxation effects of polar groups, since nanosecond time-resolved emission spectra of either retinol in glycerol or retinol bound to RBP, failed to show any time-dependent shifts in emission maxima during the time period investigated 0 to 30 ns. The degree of rotational mobility of bound retinol was investigated by time emission anisotropy measurements. The observed rotational correlation time (theta = 7.2 ns) is consistent with a rigid compact macromolecule of 21,000 molecular weight.     

The slow folding reaction of barstar: the core tryptophan region attains tight packing before substantial secondary and tertiary structure formation and final compaction of the polypeptide chain

The slow folding of a single tryptophan-containing mutant of barstar has been studied in the presence of 2 M urea at 10 degrees C, using steady state and time-resolved fluorescence methods and far and near-UV CD measurements. The protein folds in two major phases: a fast phase, which is lost in the dead time of measurement during which the polypeptide collapses to a compact form, is followed by a slow observable phase. During the fast phase, the rotational correlation time of Trp53 increases from 2.2 ns to 7.2 ns, and its mean fluorescence lifetime increases from 2.3 ns to 3.4 ns. The fractional changes in steady-state fluorescence, far-UV CD, and near-UV CD signals, which are associated with the fast phase are, respectively, 36 %, 46 %, and 16 %. The product of the fast phase can bind the hydrophobic dye ANS. These observations together suggest that the folding intermediate accumulated at the end of the fast phase has: (a) about 20 % of the native-state secondary structure, (b) marginally formed or disordered tertiary structure, (c) a water-intruded and mobile protein interior; and (d) solvent-accessible patches of hydrophobic groups. Measurements of the anisotropy decay of Trp53 suggest that it undergoes two types of rotational motion in the intermediate: (i) fast (tau(r) approximately 1 ns) local motion of its indole side-chain, and (ii) a slower (tau(r) approximately 7.2 ns) motion corresponding to global tumbling of the entire protein molecule. The ability of the Trp53 side-chain to undergo fast local motion in the intermediate, but not in the fully folded protein where it is completely buried in the hydrophobic core, suggests that the core of the intermediate is still poorly packed. The global tumbling time of the fully folded protein is faster at 5.6 ns, suggesting that the volume of the intermediate is 25 % more than that of the fully folded protein. The rate of folding of this intermediate to the native state, measured by steady-state fluorescence, far-UV CD, and near-UV CD, is 0.07(+/-0.01) min(-1) This rate compares to a rate of folding of 0.03(+/-0.005) min(-1), determined by double-jump experiments which monitor directly formation of native protein; and to a rate of folding of 0.05 min(-1), when determined from time-resolved anisotropy measurements of the long rotational correlation time, which relaxes from an initial value of 7.2 ns to a final value of 5. 6 ns as the protein folds. On the other hand, the amplitude of the short correlation time decreases rapidly with a rate of 0.24(+/-0.06) min(-1). These results suggest that tight packing of residues in the hydrophobic core occurs relatively early during the observable slow folding reaction, before substantial secondary and tertiary structure formation and before final compaction of the protein.     

Rotational modes of Ca2+-liganded calmodulin, as determined by time-domain fluorescence

The time decay of fluorescence anisotropy was monitored as a function of pH and temperature for complexes of 2,6-toluidinylnaphthalenesulfonate with calmodulin, with its proteolytic fragments, and with the 1:1 complex of calmodulin and melittin. For all the conditions examined the anisotropy decay of native calmodulin involved at least two rotational modes. These corresponded to a short correlation time of 2-3 ns, reflecting a localized motion in the vicinity of the binding site and a longer correlation time which arises from the rotation of a major portion of the molecule. The relative amplitudes of the two rotational modes were dependent upon temperature in the range 11-40 degrees C, the contribution of the more rapid mode increasing with temperature. The maximum immobilization of the probe occurred at pH 5.0 and 12 degrees C. While these results indicate the presence of internal rotations in Ca2+-liganded calmodulin, the magnitude of the longer correlation time is consistent with the crystallographic structure.     

Dynamics of benzo[a]pyrene diol epoxide adducts in poly(dG-dC).(dG-dC) studied by synchrotron excited fluorescence polarization anisotropy decay.

Time-resolved fluorescence studies have been performed on (+)-anti-7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene adducts in double-stranded poly(dG-dC).(dG-dC). Part of the adduct population gives rise to excimer fluorescence. The heterogeneous fluorescence emission decay curves at 22 degrees C could be resolved into three components with lifetimes: 0.4 ns, 3 ns and 24 ns for the total fluorescence (monomer and excimer emission), and 0.5 ns, 5 ns and 24 ns, respectively, for excimer emission alone. The relative amplitudes for the longer lifetimes were larger for the pure excimer population than for the mixed population. The fluorescence polarization anisotropy decay curves were resolved into two components of rotational correlation times: 0.4 ns and 25 ns for the total fluorescence and 0.3 ns and 33 ns for the excimer fluorescence. We interpret the two rotational correlation times to correspond to local motion of the adduct and segmental motion of the polynucleotide, respectively.     

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.     

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.     

Oligomeric state of lipocalin-1 (LCN1) by multiangle laser light scattering and fluorescence anisotropy decay

Multiangle laser light scattering and fluorescence anisotropy decay measurements clarified the oligomeric states of native and recombinant tear lipocalin (lipocalin-1, TL). Native TL is monomeric. Recombinant TL (5-68 microM) with or without the histidine tag shows less than 7% dimer formation that is not in equilibrium with the monomeric form. Fluorescence anisotropy decay showed a correlation time of 9-10 ns for TL (10 microM-1 mM). Hydrodynamic calculations based on the crystallographic structure of a monomeric TL mutant closely concur with the observed correlation time. The solution properties calculated with HYDROPRO and SOLPRO programs from the available crystallographic structure of a monomeric TL mutant concur closely with the observed fluorescence anisotropy decay. The resulting model shows that protein topology is the major determinant of rotational correlation time and accounts for deviation from the Stokes-Einstein relation. The data challenge previous gel filtration studies to show that native TL exists predominantly as a monomer in solution rather than as a dimer. Delipidation of TL results in a formation of a complex oligomeric state (up to 25%). These findings are important as the dynamic processes in the tear film are limited by diffusional, translational as well as rotational, properties of the protein.     

Environmental modulation of M13 coat protein tryptophan fluorescence dynamics

The effects of detergent [deoxycholate (DOC) and phospholipid [dimyristoylphosphatidylcholine (DMPC)] environments on the rotational dynamics of the single tryptophan residue 26 of bacteriophage M13 coat protein have been investigated by using time-resolved single photon counting measurements of the fluorescence intensity and anisotropy decay. The total fluorescence decay of tryptophan-26 is complex but rather similar in DOC as compared to DMPC when analyzed in terms of a lifetime distribution (exponential series method). This similarity, in conjunction with the almost identical steady-state fluorescence spectra, indicates only minor differences between the tryptophan environments in DOC and DMPC. The reorientational dynamics of tryptophan-26 are dominated by slow rotation of the entire protein in both detergent and phospholipid environments. The resolved anisotropy decay in DOC can be approximated by a simple hydrodynamic model of protein/detergent micelle rotational diffusion, although the data indicative slightly greater complexity in the rotational motion. The tryptophan fluorescence anisotropy is not sensitive to protein conformational changes in DOC detected by nuclear magnetic resonance on the basis of pH independence in the range 7.5-9.1. In DMPC bilayers, restricted tryptophan motion with a correlation time of approximately 2 ns is observed together with a second very slow reorientational component. Resolution of the time constant for this slow rotation is obscured by the tryptophan fluorescence time window being too short to clearly locate its anisotropic limit. The possible contribution made by axial rotational diffusion of the protein to this slow rotational process is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence and conformational stability studies of Staphylococcus nuclease and its mutants, including the less stable nuclease-concanavalin A hybrids

We report steady-state and time-resolved fluorescence studies with the single tryptophan protein, Staphylococcus aureus A, and several of its site-directed mutants. A couple of these mutants, nuclease-conA and nuclease-conA-S28G (which are hybrid proteins containing a six amino acid beta-turn substitute from concanavalin A), are found to have a much lower thermodynamic stability than the wild type. The thermal transition temperatures for nuclease-conA and S28G are 32.8 and 30.5 degrees C, which are about 20 degrees C lower than the Tm for wild-type nuclease A. These mutant proteins also are denatured by a much lower concentration of the denaturants urea and guanidine hydrochloride. We also show that an unfolding transition in the structure of the nuclease-conA hybrids can be induced by relatively low hydrostatic pressure (approximately 700 bar). The free energy for unfolding of nuclease-conA (and nuclease-conA-S28G) is found to be only 1.4 kcal/mol (and 1.2 kcal/mol) by thermal, urea, guanidine hydrochloride, and pressure unfolding. Time-resolved fluorescence intensity and anisotropy measurements with nuclease-conA-S28G show the temperature-, urea-, and pressure-perturbed states each to have a reduced average intensity decay time and to depolarize with a rotational correlation time of approximately 1.0 ns (as compared to a rotational correlation time of 11 ns for the native form of nuclease-conA-S28G at 20 degrees C).     

The effects of ligands on the conformation of phosphoglycerate kinase: fluorescence anisotropy decay and theoretical interpretation

Horse muscle phosphoglycerate kinase (PGK) is a monomer folded into two widely distant domains. In the glycolytic pathway, this enzyme catalyzes the first reaction that produces ATP. It was suggested, by analogy with yeast hexokinase, that a hinge-bending motion may be induced by the binding of specific substrates to the protein. To analyze such a motion, or any structural changes induced by ligand binding, fluorescence anisotropy decay of tryptophan residues in free and liganded PGK was studied. At 293 K, for the free protein and the binary complex with 3-phosphoglycerate, a single correlation time of 26 ns was observed, corresponding to the rotation of the overall protein, whereas upon addition of MgADP, this correlation time decreased to 10 ns. Such a decrease cannot be merely due to a change of the protein's shape and volume. To explain this, it was suggested that the fluorescence anisotropy decay of the PGK-MgADP complex corresponded to the rotation of the only buried tryptophan (Trp 335). The rotational paths of this tryptophan, in the presence and absence of the nucleotide, were established by potential energy minimization calculations. The results indicated that MgADP induces a displacement of helix alpha-13 that decreases the rotational energy barrier of Trp 335 from 16 kcal/mol in the free protein to 8 kcal/mol in the complex.     

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.     

Analysis of time-resolved fluorescence anisotropy in lipid-protein systems

The subnanosecond fluorescence and motional dynamics of the tryptophan residue in the bacteriophage M13 coat protein incorporated within pure dioleoylphosphatidylcholine (DOPC) as well as dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) and dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayers (80/20 w/w) with various L/P ratio have been investigated. The fluorescence decay is decomposed into four components with lifetimes of about 0.5, 2.0, 4.5 and 10.0 ns, respectively. In pure DOPC and DOPC/DOPG lipid bilayers, above the phase transition temperature, the rotational diffusion of the protein molecules contributes to the depolarization and the anisotropy of tryptophan is fitted to a dual exponential function. The longer correlation time, describing the rotational diffusion of the whole protein, shortens with increasing temperature and decreasing protein aggregation number. In DMPC/DMPG lipid bilayers, below the phase transition, the rotational diffusion of the protein is slowed down such that the subnanosecond anisotropy decay of tryptophan in this system reflects only the segmental motion of the tryptophan residue. Because of a heterogeneous microenvironment, the anisotropy decay must be described by three exponentials with a constant term, containing a negative coefficient and a negative decay time constant. From such a decay, the tryptophan residue within the aggregate undergoes a more restricted motion than the one exposed to the lipids. At 20 degrees C, the order parameter of the transition moment of the isolated tryptophan is about 0.9 and that for the exposed one is about 0.5.