Development and characterization of green fluorescent protein mutants with altered lifetimes

Multiple-probe fluorescence imaging applications demand an ever-increasing number of resolvable probes, and the use of fluorophores with resolvable fluorescence lifetimes can help meet this demand. Green fluorescent protein (GFP) and its variants have been widely used in spectrally resolved multiprobe imaging, but as yet, there has not been a systematic set of mutants generated with resolvable lifetimes. Therefore, to generate such mutants, we have utilized error-prone PCR and fluorescence lifetime imaging to screen for mutants of UV-excited green fluorescent protein (GFPuv) that exhibit altered fluorescence decay lifetimes. This has resulted in the isolation of GFPuv mutants displaying at least three distinctly different lifetimes in the range of 1.9-2.8 ns. Mutation of Y145 to either histidine or cysteine was found to shift the fluorescence lifetime of GFPuv from 3.03 +/- 0.03 to 2.78 +/- 0.05 ns for the Y145H mutant and to 2.74 +/- 0.05 ns for Y145C. Some of the shorter-lifetime mutants exhibited excitation peaks that were red-shifted relative to their maximal absorption, indicating that the mutations allowed the adoption of additional conformations relative to wtGFPuv. The utility of these mutants for applications in simultaneous imaging and quantification is shown by the ability to quantify the composition of binary mixtures in time-resolved images using a single detector channel. The application of the screening method for generating lifetime mutants of other fluorescent proteins is also discussed.     

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.     

Phase-sensitive fluorescence spectroscopy: A new method to resolve fluorescence lifetimes or emission spectra of components in a mixture of fluorophores

A novel phase fluorometric method is described which permits direct recording of individual emission spectra from a mixture of two flourescent compounds. Additionally, the lifetimes of each component may be determined by examination of the phase-sensitive fluorescence spectra. The method utilizes phase-sensitive detection of the sinusoidally modulated emission from a phase fluorometer. Resolution of the individual emission spectra in the mixture requires different fluorescence lifetimes for each components. Determination of the individual lifetime requires knowledge of the steady-state emission spectra of the components. Use of low-frequency (≈ 10 Hz) cross-correlated signals eliminates the need for high-frequency frequency (≈10⁶ Hz) phase-sensitive detection. A mixture of 2-p-toluidinyl-6-naphthalenesulfonic acid (TNS) and 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) was used to demonstrate the possibility of phase resolution of fluorophore mixture and to confirm theoretical predictions. A mixture of dibenzo[a,h]anthracene and dibenzo[c,g]carbazole was used to demonstrate that phase resolution is possible for spectra which overlap strongly and which are highly structured. In addition, the possibility of using phase-sensitive emission spectra for the resolution of excited-state reactions was demonstrated with anthracene and its diethylaniline exciplex. From a sample whose steady-state emission displayed both components we directly recorded the emission spectrum of anthracene monomer and the exciplex. For all these samples the dependence of the individual intensities on the phase angle of the detector agreed precisely with that expected on the basis of the individual fluorescence lifetimes. The detector phase angles chosen for suppression of each component in the mixture also agreed with the measured lifetimes. Thus, phase-sensitive fluorescence spectra can reveal individual spectral distributions or lifetimes. This method will be useful in the analysis fluorescence emissions which frequently occur from proteins, membranes and other biological samples.     

Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.     

Time-resolved fluorescence studies of tomaymycin bonding to synthetic DNAs.

Tomaymycin reacts covalently with guanine in the DNA minor groove, exhibiting considerable specificity for the flanking bases. The sequence dependence of tomaymycin bonding to DNA was investigated in synthetic DNA oligomers and polymers. The maximum extent of bonding to DNA is greater for homopurine and natural DNA sequences than for alternating purine-pyrimidine sequences. Saturation of DNA with tomaymycin has little effect on the melting temperature in the absence of unbound drug. Fluorescence lifetimes were measured for DNA adducts at seven of the ten unique trinucleotide bonding sites. Most of the adducts had two fluorescence lifetimes, representing two of the four possible binding modes. The lifetimes cluster around 2-3 ns and 5-7 ns; the longer lifetime is the major component for most bonding sites. The two lifetime classes were assigned to R and S diastereomeric adducts by comparison with previous NMR results for oligomer adducts. The lifetime difference between binding modes is interpreted in terms of an anomeric effect on the excited-state proton transfer reaction that quenches tomaymycin fluorescence. Bonding kinetics of polymer adducts were monitored by fluorescence lifetime measurements. Rates of adduct formation vary by two orders of magnitude with poly(dA-dG).poly(dC-dT), reacting the fastest at 4 x 10(-2) M-1 s-1. The sequence specificity of tomaymycin is discussed in light of these findings and other reports in the literature.     

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 lifetime: Beating the IRF and interpulse window

Fluorescence lifetime imaging captures the spatial distribution of chemical species across cellular environments employing pulsed illumination confocal setups. However, quantitative interpretation of lifetime data continues to face critical challenges. For instance, fluorescent species with known in vitro excited-state lifetimes may split into multiple species with unique lifetimes when introduced into complex living environments. What is more, mixtures of species, which may be both endogenous and introduced into the sample, may exhibit 1) very similar lifetimes as well as 2) wide ranges of lifetimes including lifetimes shorter than the instrumental response function or whose duration may be long enough to be comparable to the interpulse window. By contrast, existing methods of analysis are optimized for well-separated and intermediate lifetimes. Here, we broaden the applicability of fluorescence lifetime analysis by simultaneously treating unknown mixtures of arbitrary lifetimes—outside the intermediate, Goldilocks, zone—for data drawn from a single confocal spot leveraging the tools of Bayesian nonparametrics (BNP). We benchmark our algorithm, termed BNP lifetime analysis, using a range of synthetic and experimental data. Moreover, we show that the BNP lifetime analysis method can distinguish and deduce lifetimes using photon counts as small as 500.     

Analysis of excited-state processes by phase-modulation fluorescence spectroscopy

Fluorescence phase shift and demodulation methods were used in the analysis of excited-state reactions and to investigate solvent relaxation around fluorophores in viscous solvents. The chosen samples illustrate the results expected for fluorophores bound to biological macromolecules. These moderately simple samples served to test the theoretical predictions described in the preceding paper (J.R. Lakowicz and A.B. Balter, Biophys. Chem. 16 (1982) 99.) and to illustrate the characteristic features of phase-modulation data expected from samples which display time-dependent spectral shifts. The excited-stale protonation of acridine and exciplex formation between anthracene and diethylaniline provided examples of one-step reactions in which the lifetimes of the initially excited and the reacted species were independent of emission wavelength. Using these samples we demonstrated the following: (I) Wavelength-dependent phase shift and demodulation values can be used to prove the occurrence of an excited-state process. Proof is obtained by observation of phase angles (φ) larger than 90° or by measurement of ratios of m/cos φ > 1, where m is the modulation of the emission relative to that of the excitation. (2) For a two-state process the individual emission spectra of each state can be calculated from the wavelength-dependent phase and modulation data. (3) The phase difference or demodulation factor between the initially excited and the reacted states reveals directly the fluorescence lifetime of the product of the reaction. (4) Phase-sensitive detection of fluorescence can be used to prove that the lifetimes of both the initially excited and the reacted states are independent of emission wavelength. (5) If steady-state spectra of the individual species are known, then phase-sensitive emission spectra can be used to measure the lifetimes of the individual components irrespective of the extent of spectral overtap. (6) Spectral regions of constant lifetime can be identified by the ratios of phase-sensitive emission spectra. In addition, we examined 6-propionyl-2-dimethylaminonaphthalene(PRODAN) and N-acetyl-l-tryptophanamide (NATA) in viscous solvents where the solvent relaxation times were comparable to the fluorescence lifetimes. Using PRODAN in n-butanol we used m/cos φ measurements, relative to the blue edge of the emission, to demonstrate that solvent relaxation requires more than a single step. For NATA in propylene glycol we used phase-sensitive detection of fluorescence to directly record the emission spectra of the initially excited and the solvent relaxed states. These measurements can be easily extended to fluorophores which are bound to proteins and membranes and are likely to be useful in studies of the dynamic properties of biopolymers.     

Interpretation of fluorescence decays using a power-like model

A power-like decay function, characterized by the mean excited-state lifetime and relative variance of lifetime fluctuation around the mean value, was applied in analysis of fluorescence decays measured with the aid of time-correlated single photon counting. We have examined the fluorescence decay, in neutral aqueous medium, of tyrosine (L-tyrosine and N-acetyl-L-tyrosinamide), and of the tyrosine residues in a tryptophan-free protein, the enzyme purine nucleoside phosphorylase from Escherichia coli in a complex with formycin A (an inhibitor), and orthophosphate (a co-substrate). Tryptophan fluorescence decay was examined in neutral aqueous medium for L-tryptophan, N-acetyl-L-tryptophanamide, and for two tryptophan residues in horse liver alcohol dehydrogenase. To detect solvent effect, fluorescence decay of Nz-acetyl-L-tryptophanamide in aqueous medium was compared with that in dioxan. Hitherto, complex fluorescence decays have usually been analyzed with the aid of a multiexponential model, but interpretation of the individual exponential terms (i.e., pre-exponential amplitudes and fluorescence lifetimes), has not been adequately characterized. In such cases the intensity decays were also analyzed in terms of the lifetime distribution as a consequence of an interaction of fluorophore with environment. We show that the power-like decay function, which can be directly obtained from the gamma distribution of fluorescence lifetimes, is simpler and provides good fits to highly complex fluorescence decays as well as to a purely single-exponential decay. Possible interpretation of the power-like model is discussed.     

Theory of phase-modulation fluorescence spectroscopy for excited-state processes

Theory is presented for the analysis of excited-state reactions by fluorescence phase shift and demodulation methods. Initially, a two-state model with spectral overlap is considered to illustrate most simply the effects of excited-state reactions on the expected phase and modulation values. Secondly, a multistate model is described to illustrate the probable effects of a fluorophore interacting with several solvent molecules. We note the following unique features of phase-modulation data expected from a fluorophore whose emission spectrum shifts during the lifetime of the excited state: (1) The modulation frequency dependence of the apparent phase (τ^p) and modulation (τ^m) lifetimes of the reacted species is opposite to that of a heterogeneous population of fluorophores. (2) For the reacted species τ^p > τ^m. For a heterogeneous sample τ^p < τ^m. (3) The phase angle of the reacted species can exceed 90°. For a heterogeneous sample phase angles are always less than 90°. Thus, phase and modulation measurements can distinguish between time-dependent processes and spectral heterogeneity by observation of any feature described above. Additionally: (4) The lifetime of the product species can be measured directly. (5) Reverse relaxation can be identified, and the reverse relaxation rates calculated. (6) The wavelength-dependent phase and modulation data can be used to resolve the individual spectra from a two-state reaction. (7) And finally, under favorable conditions, a two-state excited-stale process can be distinguished from a continuous multiple-state process. In each instance, model calculations are presented to illustrate the unique potentials of phase-modulation fluorometry for investigations of excited-state processes.     

Phenylalanine-to-tyrosine singlet energy transfer in the archaebacterial histone-like protein HTa

The Archaebacterium Thermoplasma acidophilum has a histone-like protein (HTa) abundantly associated with its deoxyribonucleic acid. Each native tetrameric complex of HTa contains 20 phenylalanine residues, 4 tyrosine residues, and no tryptophan. When the protein was excited by radiation at 252 nm, which is a wavelength absorbed predominantly by phenylalanine, the fluorescent emission was mostly from tyrosine. According to the excitation spectrum for this tyrosine fluorescence, the cause was energy transfer from phenylalanine, which occurred with about 50% efficiency. When the tyrosine residues were removed enzymatically, the excited-state lifetime of the phenylalanine residues nearly doubled. Because of energy transfer, the tyrosine emission had two apparent fluorescence decay lifetimes; one lifetime (3.9 ns) was that of tyrosine while the second (12.1 ns) corresponded to the excited state of phenylalanine.     

Conformational effects on tryptophan fluorescence in cyclic hexapeptides

The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.     

Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy

O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation. We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case. Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity. At least two components were detected, with lifetimes of approximately 2.5 and 0.5 ns. The lifetimes are very close to the values measured in bulk solutions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited state.     

Heterogeneity of the two tryptophanyl residues in the lac repressor of Escherichia coli.

The wild-type lac repressor of Escherichia coli is a tetrameric protein which contains two tryptophanyl residues per subunit at positions 190 and 209. Solute perturbation studies of the tryptophan fluorescence of the repressor were performed using a polar but uncharged quencher, acrylamide, to prevent possible bias caused by ionic quenchers. The results indicate that the two tryptophan residues have different accessibilities to the quencher. In addition, contrary to a previous report, the accessibility of these tryptophan residues is not altered by isopropyl-β-d-thiogalactoside (IPTG) binding to the repressor. Similar studies with mutant lac repressor containing only a single tryptophan either at positions 190 or 209 suggest that tryptophan 209 is located in a region which is perturbed by inducer binding. That the two tryptophanyl residues have heterogeneous environments was further confirmed by nanosecond fluorescence spectroscopy which showed the wild-type lac repressor exhibiting two excited-state lifetimes, τ₁ = 5.3 ns and τ₂ = 10 ns. In the presence of 10^?3m IPTG, only a single lifetime of 6 ns was observed for the wild-type repressor suggesting that the inducer perturbs the tryptophan residue with the longer lifetime but not the one with the shorter lifetime. This is in accord with the observation that the mutant repressor containing only tryptophan 190 (the Tyr-209 repressor) has a single lifetime of 4.5 ns which is not altered by IPTG binding. The surprising finding that the mutant repressor which contains only tryptophan 209 (the Tyr-190 repressor) shows two excited-state lifetimes has been interpreted to indicate that the repressor either does not exhibit fourfold symmetry in its subunit arrangement or is present in two different conformational states.     

Two-Photon Lifetime Imaging of Voltage Indicating Proteins as a Probe of Absolute Membrane Voltage

Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage in a manner that is insensitive to the protein expression level, illumination intensity, or photon detection efficiency. First, we tested several GEVIs for 2P brightness, response speed, and voltage sensitivity. ASAP1 and a previously described citrine-Arch electrochromic Förster resonance energy transfer sensor (dubbed CAESR) showed the best characteristics. We then characterized the voltage-dependent lifetime of ASAP1, CAESR, and ArcLight under voltage-clamp conditions. ASAP1 and CAESR showed voltage-dependent lifetimes, whereas ArcLight did not. These results establish 2P fluorescence lifetime imaging as a viable means of measuring absolute membrane voltage. We discuss the prospects and improvements necessary for applications in tissue.     

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.     

Redox effects on the excited-state lifetime in chlorosomes and bacteriochlorophyll c oligomers.

Oligomers of [E,E] BChl CF (8, 12-diethyl bacteriochlorophyll c esterified with farnesol (F)) and [Pr,E] BChl CF (analogously, M methyl, Pr propyl) in hexane and aqueous detergent or lipid micelles were studied by means of steady-state absorption, time-resolved fluorescence, and electron spin resonance spectroscopy. The maximum absorption wavelength, excited-state dynamics, and electron spin resonance (EPR) linewidths are similar to those of native and reconstituted chlorosomes of Chlorobium tepidum. The maximum absorption wavelength of oligomers of [E,E] BChl CF was consistently blue-shifted as compared to that of [Pr,E] BChl CF oligomers, which is ascribed to the formation of smaller oligomers with [E,E] BChl CF than [Pr,E] BChl CF. Time-resolved fluorescence measurements show an excited-state lifetime of 10 ps or less in nonreduced samples of native and reconstituted chlorosomes of Chlorobium tepidum. Under reduced conditions the excited-state lifetime increased to tens of picoseconds, and energy transfer to BChl a or long-wavelength absorbing BChl c was observed. Oligomers of [E,E] BChl CF and [Pr,E] BChl CF in aqueous detergent or lipid micelles show a similar short excited-state lifetime under nonreduced conditions and an increase up to several tens of picoseconds upon reduction. These results indicate rapid quenching of excitation energy in nonreduced samples of chlorosomes and aqueous BChl c oligomers. EPR spectroscopy shows that traces of oxidized BChl c radicals are present in nonreduced and absent in reduced samples of chlorosomes and BChl c oligomers. This suggests that the observed short excited-state lifetimes in nonreduced samples of chlorosomes and BChl c oligomers may be ascribed to excited-state quenching by BChl c radicals. The narrow EPR linewidth suggests that the BChl c are arranged in clusters of 16 and 6 molecules in chlorosomes of Chlorobium tepidum and Chloroflexus aurantiacus, respectively.     

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.     

An unusual red-edge excitation and time-dependent Stokes shift in the single tryptophan mutant protein DD-carboxypeptidase from Streptomyces: the role of dynamics and tryptophan rotamers

The fluorescence emission of the single tryptophan (W233) of the mutant protein DD-carboxypeptidase from streptomyces is characterized by a red-edge excitation shift (REES), i.e., the phenomenon that the wavelength of maximum emission depends on the excitation wavelength. This phenomenon is an indication for a strongly reduced dynamic environment of the single tryptophan, which has a very low accessibility to the solvent. The REES shows, however, an unusual temperature and time dependence. This, together with the fluorescence lifetime analysis, showing three resolvable lifetimes, can be explained by the presence of three rotameric states that can be identified using the Dead-End Elimination method. The three individual lifetimes increase with increasing emission wavelength, indicating the presence of restricted protein dynamics within the rotameric states. This is confirmed by time-resolved anisotropy measurements that show dynamics within the rotamers but not among the rotamers. The global picture is that of a protein with a single buried tryptophan showing strongly restricted dynamics within three distinct rotameric states with different emission spectra and an anisotropic environment.     

Tryptophanyl fluorescence lifetime distribution of hyperthermophilic beta-glycosidase from molecular dynamics simulation: a comparison with the experimental data

A molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for beta-glycosidase from the hyperthermophilic bacterium Solfolobus sulfotaricus (Sbeta gly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of Sbeta gly results from a bimodal distribution of fluorescence lifetimes with a short-lived component centered at 2.5 ns and a long-lived one at 7.4 ns (Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71-79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern-Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of Sbeta gly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition Sbeta gly evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side-chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results.