A fluorescence lifetime study of virginiamycin S using multifrequency phase fluorometry

Using multifrequency phase fluorometry, fluorescence lifetimes have been assigned to the different protolytic forms of the antibiotic virginiamycin S. These lifetimes are 0.476 +/- 0.005 ns for the uncharged form, 1.28 +/- 0.2 and 7.4 +/- 0.2 ns for the zwitterionic form, 1.19 +/- 0.01 ns for the negatively charged form, and 1.9 +/- 0.1 ns for the double negatively charged form. The assignments are based on lifetime measurements as a function of pH, volume percent ethanol, and excitation wavelength. Excited-state proton transfer is taken into account. It is complete at pH values lower than 1, and no fluorescence of the fully protonated charged form is observed. At pH 8, an excited-state pK* increase is calculated, but proton association is too slow to cause excited-state proton transfer. The addition of divalent cations, at pH 9.4, increases the lifetime of the negatively charged form to a value dependent upon the specific nature of the cation (7.58 +/- 0.06 ns for Mg2+, 6.54 +/- 0.02 ns for Ca2+, and 3.74 +/- 0.05 ns for Ba2+). Monovalent cations do not influence the lifetimes, indicating that their binding to the macrocycle does not influence the fluorescent moiety. The model compound 3-hydroxypicolinamide shows an analogous behavior, but the retrieved lifetime can differ significantly.     

Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression

BACKGROUND: Frequency-domain fluorescence lifetime imaging microscopy (FLIM) is finding increasing use in the analysis of biological systems. However, the calibration, determination of resolvable lifetime differences, and evaluation of artifacts have not been extensively treated. We describe a multi-point method for calibrating a frequency-domain FLIM system, characterize the minimum detectable heterogeneity and intra- and inter-image lifetime differences, discuss the statistical treatment of FLIM data, and suggest methods for minimizing artifacts. METHODS: A set of solutions exhibiting single-component lifetimes suffice for accurately calibrating a reference material with a single-component lifetime, even in the absence of accurate data on the lifetimes of the individual solutions or the reference material. We used a set of rhodamine 6G solutions quenched with varying concentrations of iodide, leading to lifetimes of 0.5--4.0 ns, to calibrate a 1 microM reference solution of rhodamine 6G in water. RESULTS: We measured a value of 4.11 ns with an estimated absolute error of +/-0.05 ns for the rhodamine 6G reference solution. With 57.7 MHz modulation, the minimum detectable inter-image lifetime difference was 0.1--0.15 ns and the minimum detectable intra-image lifetime difference was 4--5 ps, allowing solutions differing in lifetime by 40 and 70 ps to be easily distinguished. The minimum detectable lifetime heterogeneity was 50--80 ps. Evaluation of replicate measurements of single solutions demonstrated that inter-image instrument errors exceeded those predicted from intra-image statistics by more than an order of magnitude. We also measured lifetimes and heterogeneity in 4 GFP variants (WTGFP, EGFP, S65T, and EYFP) with the technique. CONCLUSION: The multi-point calibration method is applicable to any system consisting of single-component lifetimes. Applying the method in our FLIM microscope allowed us to demonstrate a previously unreported degree of lifetime resolution in a FLIM microscope. Cytometry 43:248-260;2001.     

Determination of the excited-state lifetimes of the tryptophan residues in barnase, via multifrequency phase fluorometry of tryptophan mutants.

A multifrequency phase fluorometric study is described for wild-type barnase and engineered mutant proteins in which tryptophan residues have been replaced by less fluorescent residues which do not interfere with the determination of the tryptophan emission spectra and lifetimes. The lifetimes of the three tryptophans in the wild-type protein have been resolved. Trp-35 has a single fluorescence lifetime, which varies in the different proteins between 4.3 and 4.8 ns and is pH-independent between pH 5.8 and 8.9. Trp-71 and Trp-94 behave as an energy-transfer couple with both forward and reverse energy transfer. The couple shows two fluorescence lifetimes: 2.42 (+/-0.2) and 0.74 (+/-0.1) ns at pH 8.9, and 0.89 (+/-0.05) and 0.65 (+/-0.05) ns at pH 5.8. In the mutant Trp-94----Phe the lifetime of Trp-71 is 4.73 (+/-0.008) ns at high pH and 4.70 (+/-0.004) ns at low pH. In the mutant Trp-71----Tyr, the lifetime of Trp-94 is 1.57 (+/-0.01) ns at high pH and 0.82 (+/-0.025) ns at low pH. From these lifetimes, one-way energy-transfer efficiencies can be calculated according to Porter [Porter, G.B. (1972) Theor. Chim. Acta 24, 265-270]. At pH 8.9, a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was 86% for forward and 4% for reverse transfer (all +/-2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of F?rster [F?rster, T. (1948) Ann. Phys. (Leipzig) 2, 55-75].(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell

Fluorescence lifetime imaging microscopy (FLIM) is a technique in which the mean fluorescence lifetime of a chromophore is measured at each spatially resolvable element of a microscope image. The nanosecond excited-state lifetime is independent of probe concentration or light path length but dependent upon excited-state reactions such as fluorescence resonance energy transfer (FRET). These properties of fluorescence lifetimes allow exploration of the molecular environment of labelled macromolecules in the interior of cells. Imaging of fluorescence lifetimes enables biochemical reactions to be followed at each microscopically resolvable location within the cell.     

In vivo resolution of multiexponential decays of multiple near-infrared molecular probes by fluorescence lifetime-gated whole-body time-resolved diffuse optical imaging

The biodistribution of two near-infrared fluorescent agents was assessed in vivo by time-resolved diffuse optical imaging. Bacteriochlorophyll a (BC) and cypate-glysine-arginine-aspartic acid-serine-proline-lysine-OH (Cyp-GRD) were administered separately or combined to mice with subcutaneous xenografts of human breast adenocarcinoma and slow-release estradiol pellets for improved tumor growth. The same excitation (780 nm) and emission (830 nm) wavelengths were used to image the distinct fluorescence lifetime distribution of the fluorescent molecular probes in the mouse cancer model. Fluorescence intensity and lifetime maps were reconstructed after raster-scanning whole-body regions of interest by time-correlated single-photon counting. Each captured temporal point-spread function (TPSF) was deconvolved using both a single and a multiexponental decay model to best determine the measured fluorescence lifetimes. The relative signal from each fluorophore was estimated for any region of interest included in the scanned area. Deconvolution of the individual TPSFs from whole-body fluorescence intensity scans provided corresponding lifetime images for comparing individual component biodistribution. In vivo fluorescence lifetimes were determined to be 0.8 ns (Cyp-GRD) and 2 ns (BC). This study demonstrates that the relative biodistribution of individual fluorophores with similar spectral characteristics can be compartmentalized by using the time-domain fluorescence lifetime gating method.     

Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy.

BACKGROUND: Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long acquisition times and more photo bleaching. An alternative is spinning-disc confocal whereby samples are scanned simultaneously by thousands of pinholes, resulting in a virtually instantaneous image with more than tenfold reduced photo bleaching. METHODS: A spinning disc unit was integrated into an existing FLIM system. Measurements were made of fluorescent beads with a lifetime of 2.2 ns against a 5.3 ns fluorescent background outside the focal plane. In addition, living HeLa cells were imaged with different lifetimes in the cytosol and the plasma membrane. RESULTS: In spinning-disc mode, a lifetime of the beads of 2.8 ns was measured, whereas in wide field a lifetime of 4.1 ns was measured. Lifetime contrast within living HeLa cells could be resolved with the spinning-disc unit, where this was impossible in wide field. CONCLUSIONS: Integration of a spinning-disc unit into a frequency-domain FLIM instrument considerably reduces artifacts, while maintaining the advantages of wide field. For FLIM on objects with 3D lifetime structure, spinning-disc is by far preferable over wide-field measurements.     

Picosecond-resolved fluorescence spectra of D-amino-acid oxidase. A new fluorescent species of the coenzyme

Protein dynamics of D-amino-acid oxidase in the picosecond region was investigated by measuring time-resolved fluorescence of the bound coenzyme, FAD. The observed nonexponential fluorescence decay curves were analyzed with four-exponential decay functions. The fluorescence lifetimes at the best fit were 26.6 +/- 0.7 ps, 44.0 +/- 4.2 ps, 177 +/- 11 ps, and 2.28 +/- 0.21 ns at 20 degrees C and 25.2 +/- 3.0 ps, 50.3 +/- 8.7 ps, 228 +/- 27 ps, and 2.75 +/- 0.33 ns at 5 degrees C. Component fractions with the shortest lifetime, ca. 26 ps, were always negative and close to -1. The other fluorescent components of the lifetimes, ca. 47 ps, 200 ps, and 2.6 ns, with positive fractions were assigned to different forms of the enzyme including the dimer, the monomer, and free FAD dissociated from the enzyme. Measurements of the time-resolved fluorescence spectra revealed that the maximum wavelengths of the spectra shifted toward shorter wavelength by 65 nm at 20 degrees C and 36 nm at 5 degrees C within 100 ps after pulsed excitation. The remarkable blue shift was not observed in free FAD. The first spectra immediately after the excitation of the enzyme exhibited maximum wavelengths of 584 nm at 20 degrees C and 557 nm at 5 degrees C. The fluorescence spectra obtained at times later than 100 ps are in good agreement with the one obtained under steady-state excitation of D-amino-acid oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Determination of the topography of cytochrome b5 in lipid vesicles by fluorescence quenching

Cytochrome b5, a protein isolated from the endoplasmic reticulum by detergent extraction, interacts spontaneously with small unilamellar phosphatidylcholine vesicles. When the vesicles are made from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), the tryptophan fluorescence of the cytochrome is enhanced, and when they are made from 1-palmitoyl-2-(dibromostearoyl) phosphatidylcholine (BRPC), the fluorescence is quenched. A series of BRPC were synthesized with bromine atoms at the 6,7, 9,10, 11,12 or 15,16 positions. The vesicles synthesized from each of these lipids were similar in size to those made from POPC. The relative fluorescence intensities of the cytochrome b5 in POPC and 6,7-, 9,10-, 11,12- and 15,16- BRPC were 100, 19.4, 29.4, 37.1, and 54.0, respectively. These data suggest that the exposed tryptophan(s) is (are) at a depth of 0.7 nm below the surface of the vesicle. Bromine is a collisional quencher; hence, these data may indicate the relative position of the lipid annulus around the protein rather than the depth of the protein below the average vesicle surface. Cytochrome b5 contains three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three potentially fluorescent tryptophans, and determinations of fluorescent quantum yield indicate all three are fluorescent with an average quantum yield, when in POPC vesicles, of 0.21. Fluorescence lifetime measurements by the demodulation technique indicated heterogeneity of fluorescence lifetimes in all vesicles. The lifetimes in the BRPC vesicles ranged from 2.0 to 2.4 ns compared to a value of 3.3 ns in POPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal characteristics of recombinant green fluorescent protein (GFPuv) extracted from Escherichia coli

AIMS: The thermal stability of isolated and extracted recombinant green fluorescent protein (GFPuv) was evaluated by analysing the loss of fluorescence intensity. METHODS AND RESULTS: GFPuv was expressed by Escherichia coli, extracted by the three-phase partitioning method and purified by elution through an hydrophobic interaction column. The collected fractions were further diluted in Tris-HCl-EDTA (pH 8.0) and subjected to continuous heating at set temperatures (45-95 degrees C). From a standard curve relating fluorescence intensity to GFPuv concentration, the loss of fluorescence intensity was converted to denatured GFPuv concentration (microg ml-1). To determine the extent of the thermal stability of GFPuv, decimal reduction times (D-values), z-value and energy of activation (Ea) were calculated. CONCLUSIONS: For temperatures between 45 and 70 degrees C, extracted native GFPuv activity decreased from 11 to 75% relative to initial native protein concentration above 70 degrees C, the average decrease in GFPuv fluorescence was between 72 to 83%. SIGNIFICANCE AND IMPACT OF THE STUDY: The thermal stability of GFPuv provides the basis for its potential utility as a fluorescent biological indicator to assess the efficacy of the treatment of liquids and materials exposed to steam.     

Fluorescent proteins for single‐molecule fluorescence applications

We present single‐molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single‐molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single‐molecule applications, demonstrated by high photostability and rare fluorescence‐intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single‐molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Investigation of the structural determinants of the intrinsic fluorescence emission of the trp repressor using single tryptophan mutants.

The fluorescence decay properties of wild-type trp repressor (TR) have been characterized by carrying out a multi-emission wavelength study of the frequency response profiles. The decay is best analyzed in terms of a single exponential decay near 0.5 ns and a distribution of lifetimes centered near 3-4 ns. By comparing the recovered decay associated spectra and lifetime values with the structure of the repressor, tentative assignments of the two decay components recovered from the analysis to the two tryptophan residues, W19 and W99, of the protein have been made. These assignments consist of linking the short, red emitting component to emission from W99 and most of the longer bluer emitting lifetime distribution to emission from W19. Next, single tryptophan mutants of the repressor in which one of each of the tryptophan residues was substituted by phenylalanine were used to confirm the preliminary assignments, inasmuch as the 0.5-ns component is clearly due to emission from tryptophan 99, and much of the decay responsible for the recovered distribution emanates from tryptophan 19. The data demonstrate, however, that the decay of the wild-type protein is not completely resolvable due both to the large number of components in the wild-type emission (at least five) as well as to the fact that three of the five lifetime components are very close in value. The fluorescence decay of the wild-type decay is well described as a combination of the components found in each of the mutants. However, whereas the linear combination analysis of the 15 data sets (5 from the wild-type and each mutant) yields a good fit for the components recovered previously for the two mutants, the amplitudes of these components in the wild-type are not recovered in the expected ratios. Because of the dominance of the blue shifted emission in the wild-type protein, it is most likely that subtle structural differences in the wild-type as compared with the mutants, rather than energy transfer from tryptophan 19 to 99, are responsible for this failure of the linear combination hypothesis.     

Discrimination of DNA and RNA in cells by a vital fluorescent probe: lifetime imaging of SYTO13 in healthy and apoptotic cells

BACKGROUND: Of the few vital DNA and RNA probes, the SYTO dyes are the most specific for nucleic acids. However, they show no spectral contrast upon DNA or RNA binding. We show that fluorescence lifetime imaging using two-photon excitation of SYTO13 allows differential and simultaneous imaging of DNA and RNA in living cells, as well as sequential and repetitive assessment of staining patterns. METHODS: Two-photon imaging of SYTO13 is combined with lifetime contrast, using time-gated detection. We focus on distinguishing DNA and RNA in healthy and apoptotic Chinese hamster ovary cells. RESULTS: In healthy cells, SYTO13 has a fluorescence lifetime of 3.4 +/- 0.2 ns when associated with nuclear DNA. Bound to RNA, its lifetime is 4.1 +/- 0.1 ns. After induction of apoptosis, clusters of SYTO13 with fluorescence lifetime of 3.4 +/- 0.2 ns become apparent in the cytoplasm. They are identified as mitochondrial DNA on the basis of colocalization experiments with the DNA-specific dye, DRAQ5, and the mitochondrial-specific dye, CMXRos. Upon progression of apoptosis, the lifetime of SYTO13 attached to DNA shortens significantly, which is indicative of changes in the molecular environment of the dye. CONCLUSIONS: We have characterized SYTO13 as a vital lifetime probe, allowing repetitive and differential imaging of DNA and RNA.     

Distribution of mitochondrial NADH fluorescence lifetimes: steady-state kinetics of matrix NADH interactions

The lifetimes of fluorescent components of matrix NADH in isolated porcine heart mitochondria were investigated using time-resolved fluorescence spectroscopy. Three distinct lifetimes of fluorescence were resolved: 0.4 (63%), 1.8 (30%), and 5.7 (7%) ns (% total NADH). The 0.4 ns lifetime and the emission wavelength of the short component were consistent with free NADH. In addition to their longer lifetimes, the remaining pools also had a blue-shifted emission spectrum consistent with immobilized NADH. On the basis of emission frequency and lifetime data, the immobilized pools contributed >80% of NADH fluorescence. The steady-state kinetics of NADH entering the immobilized pools was measured in intact mitochondria and in isolated mitochondrial membranes. The apparent binding constants (K(D)s) for NADH in intact mitochondria, 2.8 mM (1.9 ns pool) and >3 mM (5.7 ns pool), were on the order of the estimated matrix [NADH] (approximately 3.5 mM). The affinities and fluorescence lifetimes resulted in an essentially linear relationship between matrix [NADH] and NADH fluorescence intensity. Mitochondrial membranes had shorter emission lifetimes in the immobilized poo1s [1 ns (34%) and 4.1 ns (8%)] with much higher apparent K(D)s of 100 microM and 20 microM, respectively. The source of the stronger NADH binding affinity in membranes is unknown but could be related to high order structure or other cofactors that are diluted out in the membrane preparation. In both preparations, the rate of NADH oxidation was proportional to the amount of NADH in the long lifetime pools, suggesting that a significant fraction of the bound NADH might be associated with oxidative phosphorylation, potentially in complex 1.     

Global analysis of fluorescence lifetime imaging microscopy data

Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.     

Use of a fluorescence lifetime imaging microscope in an apoptosis assay of Ewing's sarcoma cells with a vital fluorescent probe

A fluorescence lifetime imaging microscope (FLIM) was applied to study early-stage apoptotic cells stained with a SYTO13 dye. The fluorescence lifetime of SYTO13 in healthy cells was 3.8+/-0.3ns but was reduced to 2.4+/-0.4 and 1.9+/-0.2ns after a 3-h period of incubation with SYTO13 when doxorubicin, a known inducer of apoptosis, was added to human Ewing's family tumor cells at final concentrations of 250 and 500nM, respectively, in a dose-dependent experiment. On the other hand, in a time-dependent experiment, the fluorescence lifetime decreased to 2.5+/-0.5 and 1.7+/-0.4ns at a doxorubicin concentration of 750nM after 2 and 4h, respectively. A possible explanation for these results is self-quenching induced by a change in interprobe distance that arises from the condensation of DNA during apoptosis. In this study, the FLIM system was employed to investigate early-stage apoptosis that involves only small morphological changes, suggesting the potential advantage of this method for evaluating small biological effects in living cells.     

Time-resolved fluorescence studies of genetically engineered Escherichia coli glutamine synthetase. Effects of ATP on the tryptophan-57 loop.

Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy. The W57L mutant (retains tryptophan at residue 158) and the W158S mutant (retains tryptophan at residue 57) are both characterized by heterogeneous exponential decay kinetics. Global analysis indicates that for the Mn-bound form of the enzyme at pH 7.4 the fluorescence of both tryptophans is best described by a sum of three discrete expontials with recovered lifetimes of 4.77, 1.72, and 0.10 ns for Trp-57 and 5.04, 2.28, and 0.13 ns for Trp-158. The wild-type enzyme also exhibits decay kinetics described by a triple-exponential model with similar lifetime components. The individual tryptophans are distinguishable by the fractional intensities of the resolvable lifetimes. The wild-type and W158S enzymes are dominated by the 5-ns component which provides nearly 60% and 65%, respectively, of the fractional intensity at five wavelengths spanning the emission spectrum. In contrast, the W57L enzyme demonstrates a larger fraction of the 2-ns lifetime species (60%) and only 35% of the longer lifetime component. The substrate ATP induces a shift to approximately 90% of the 5-ns component for the wild-type and W158S enzymes, whereas the W57L protein is essentially unaffected by this ligand. Steady-state quenching studies with iodide indicate that addition of ATP results in a 3.0-3.5-fold decrease in the apparent Stern-Volmer quenching constants for the wild-type and W158S enzymes. Phase/modulation experiments at several iodide concentrations indicate that the median, 2 ns, lifetime component is selectively quenched compared to the 5-ns lifetime component. These results suggest a model where ATP binding results in a shift in the equilibrium distribution of microconformational states populated by Trp-57. ATP shifts this equilibrium nearly completely to the states exhibiting the long-lifetime component which, based on quenching studies, is less solvent-accessible than the conformational states associated with the other lifetime components.     

Time-resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: conformational analysis of intermediates and transition-state complexes.

Single tryptophan-containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency-domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn-bound wild-type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp-57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp-158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry 30, 3406-3416). In addition, Trp-57 and Trp-158 both exhibit an ATP-induced increase in the relative fraction of the long lifetime component, whereas only Trp-57 is affected by this ligand at pH 7.5. The transition-state analogue L-methionine-(R,S)-sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp-158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the gamma-glutamylphosphate-ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp-158, but has no effect on Trp-57. Similar results were obtained when ATP was added to the MSOX complex; Trp-57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp-158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX-ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp-57 loop and the central loop (Trp-158) during several intermediate stages of catalysis.     

Calcium-induced conformational change in fragment 1-86 of factor X

Time-resolved fluorescence of the single tryptophan residue Trp41 in fragment 1-86 of factor X (FX F1-86) is studied using a time-correlated single photon counting technique with synchrotron radiation as the excitation source. Calcium ions are believed to induce a conformational change in the N-termini of the activated factor X and other vitamin K dependent proteins, which is accompanied by a decrease in fluorescence intensity. The titration with calcium yields a sigmoidal fluorescence titration curve with a transition midpoint concentration of 0.44 mM. The wavelength-dependent tryptophan fluorescence decays of the apo-FX F1-86 (in the absence of calcium) and Ca-FX F1-86 are characterized by conventional multiexponential analysis and fluorescence lifetime distribution analysis. In the absence of calcium there are three significant classes of fluorescence lifetimes (ns) that are nearly wavelength independent: 0.55 +/- 0.08 (component A), 2.6 +/- 0.1 (component B), and 5.3 +/- 0.3 (component C). However, their preexponential amplitudes vary with wavelength. The decay associated emission spectra of the individual components show that components B and C contribute over 85% to the total fluorescence for all examined wavelengths. However, in the presence of calcium, the analysis of the time-resolved fluorescence data of Ca-FX F1-86 yields four wavelength-independent lifetimes (ns) of 0.30 +/- 0.09 (component D), 0.65 +/- 0.10 (component A), 2.7 +/- 0.2 (component B), and 5.4 +/- 0.3 (component C). Calcium addition to the apo-FX F1-86 leads to a decrease in the fluorescence intensities of components B and C while their decay times remain unaffected. In Ca-FX F1-86 an additional component D arises that has a decay time of 0.30 ns and that contributes up to 35% to the total fluorescence intensity. A comparison with a previous investigation of prothrombin fragment 1 demonstrates the extensive structural and functional homology between the N termini of prothrombin and factor X(a).