Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry

We report the first anisotropy decays of protein fluorescence obtained using a frequency-domain fluorometer. The ultraviolet light source (300 nm) was a ring dye laser equipped with an intracavity frequency doubler, pumped by an argon ion laser. The data, measured at modulation frequencies from 2 to 200 MHz, reveal the presence of subnanosecond motions (0.1-0.2 ns) of the single tryptophan residues in melittin and monellin. For melittin the data also indicate the presence of slower motions near 1 ns, which may be the result of concerted motions of several peptide units. Smaller amplitude motions, on a similar timescale, were observed for the single tryptophan residue in staphylococcal nuclease. We demonstrate using N-acetyl-L-tryptophanamide in water that the method of frequency-domain fluorometry is capable of measuring correlation times as short as 50 ps. This method can provide data for the direct comparison of measured anisotropy decays with those predicted from molecular dynamics calculations.     

Interpretation of in vivo fluorescence and cell division rates of natural phytoplankton using a cell cycle model

A natural population of phytoplankton was collected from theMenai Straits and incubated in a continuous culture system undernatural illumination. In vivo fluorescence data were used toderive cell cycle parameters, and the division rates of cellsin the population were analysed using a transition-point modelof the cell cycle. The results indicated that for modellingpurposes the mixed population could be regarded as being composedof a single species having properties equivalent to the averagefor the population. Fluorescence measurements were subsequcntlyrepeated on samples of the in situ population at the collectionsite, and the cell cycle model used to interpret the growthrate of the phytoplankton in the natural environment.     

Analysis of fluorescence anisotropy decays by a least square method

A method of fluorescence anisotropy decay analysis is described in this work. The transient anisotropy r(ex)(t) measured in a photocounting pulsefluorimeter is fitted by a non linear least square procedure to the ratio of convolutions of the apparatus response function g(t) by sums of appropriate exponential functions. This method takes rigorously into account the apparatus response function and is applicable to any shape of the later as well as to any values of fluorescence decay times and correlation times. The performances of the method have been tested with data simulated from measured response functions corresponding to an air lamp and a high pressure nitrogen lamp. The statistical standard errors of the anisotropy deca parameters have been found to be smaller than the standard errors previously calculated for the moment method. A systematic error delta in the fluorescence decay time entailed an error deltatheta in the correlation time such as Deltatheta/theta < deltatau/tau. By this method, good fitting of experimental data have been achieved very conveniently and accurately.     

Analysis of time-resolved fluorescence anisotropy decays.

We discuss the analysis of time-correlated single photon counting measurements of fluorescence anisotropy. Particular attention was paid to the statistical properties of the data. The methods used previously to analyze these experiments were examined and a new method was proposed in which parallel- and perpendicular-polarized fluorescence curves were fit simultaneously. The new method takes full advantage of the statistical properties of the measured curves; and, in some cases, it is shown to be more sensitive than other methods to systematic errors present in the data. Examples were presented using experimental and simulated data. The influence of fitting range on extracted parameters and statistical criteria for evaluating the quality of fits are also discussed.     

A nomogram for deconvolution of single exponential fluorescence decays.

An extremely rapid technique for deconvolving single exponential luminescence decay data is described that involves essentially no mathematical manipulation of the experimental data. The method permits "real time" measurement of deconvolved luminescence lifetimes with conventional pulsed, lifetime-fluorometers and phosphorimeters. The method assumes that the true luminescence decay of the chromophore is accurately represented by a single exponential decay function.     

Interpretation of nitrogen isotope signatures using the NIFTE model

Nitrogen cycling in forest soils has been intensively studied for many years because nitrogen is often the limiting nutrient for forest growth. Complex interactions between soil, microbes, and plants and the consequent inability to correlate δ¹⁵N changes with biologic processes have limited the use of natural abundances of nitrogen isotopes to study nitrogen (N) dynamics. During an investigation of N dynamics along the 250-year-old successional sequence in Glacier Bay, Alaska, United States, we observed several puzzling isotopic patterns, including a consistent decline in δ¹⁵N of the late successional dominant Picea at older sites, a lack of agreement between mineral N δ¹⁵N and foliar δ¹⁵N, and high isotopic signatures for mycorrhizal fungi. In order to understand the mechanisms creating these patterns, we developed a model of N dynamics and N isotopes (Nitrogen Isotope Fluxes in Terrestrial Ecosystems, NIFTE), which simulated the major transformations of the N cycle and predicted isotopic signatures of different plant species and soil pools. Comparisons with field data from five sites along the successional sequence indicated that NIFTE can duplicate observed patterns in δ¹⁵N of soil, foliage, and mineral N over time. Different scenarios that could account for the observed isotopic patterns were tested in model simulations. Possible mechanisms included increased isotopic fractionation on mineralization, fractionation during the transfer of nitrogen from mycorrhizal fungi to plants, variable fractionation on uptake by mycorrhizal fungi compared to plants, no fractionation on mycorrhizal transfer, and elimination of mycorrhizal fungi as a pool in the model. The model results suggest that fractionation during mineralization must be small (˜2‰), and that no fractionation occurs during plant or mycorrhizal uptake. A net fractionation during mycorrhizal transfer of nitrogen to vegetation provided the best fit to isotopic data on mineral N, plants, soils, and mycorrhizal fungi. The model and field results indicate that the importance of mycorrhizal fungi to N uptake is probably less under conditions of high N availability. Use of this model should encourage a more rigorous assessment of isotopic signatures in ecosystem studies and provide insights into the biologic transformations which affect those signatures. This should lead to an enhanced understanding of some of the fundamental controls on nitrogen dynamics. Received: 1 July 1998 / Accepted: 23 December 1998     

Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes

Nanosecond decays of the fluorescence anisotropy, $\text{r}$, were studied for the emission of 1,6-diphenyl-l,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes.The relative contributions of the fast and the infinitely slow decaying component to the steady-state value, $\text{r}$, of the fluorescence anisotropy were very similar for artifical and biological membranes.Angles, θ, of the cone, by which the motion of the fluorescent molecule is limited, were calculated from the intensity of the infinitely slow decaying anisotropy component and compared with steady-state fluorescence anisotropies and with ‘microviscosities’, 〈η〉. An increase in 〈η〉 from 1.5 to 5.2 P in our systems was accompanied by a decrease in θ from 49° to 30° while the decrease in the mean motional relaxation times, $\text{φ}{}_{\mathrm{<mtext>f</mtext>}}$, of the label molecule was not more than 1 ns and due mainly to changes in the potential, by which the diffusion of DPH in the membrane is restricted. From these observations we conclude that differences in the steady-state fluorescence anisotropy and in ‘microviscosities’ of cholesterol-containing membranes ($\text{r}\text{> 0.15}$) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.     

Resolution of initially excited and relaxed states of tryptophan fluorescence by differential-wavelength deconvolution of time-resolved fluorescence decays

We describe a new procedure for the analysis of time-resolved decays of fluorescence intensity. This procedure was used to resolve the emission spectra of the initially excited and solvent relaxed states of a tryptophan derivative in viscous solution. Specifically, we examined N-acetyl-l-tryptophanamide (AcTrpNH₂) in viscous and nonviscous solutions of propylene glycol. Time-resolved decays of fluorescence intensity were collected at wavelengths across the emission spectra. Instead of the usual procedure of deconvolving these data with the time profile of the exciting pulse, we deconvolved these data using the response observed on the short-wavelength side of the emission. If one assumes that this emission results only from the initially excited state (F), then the nonzero decay time calculated using deconvolution is that of the solvent relaxed state (R). For our specific case of AcTrpNH₂ the emission spectra of the F and R states overlap at most wavelengths longer than the short-wavelength side of the emission (310 nm). As a result, differential-wavelength deconvolution yields two lifetimes and amplitudes, one pair representing the relaxed state and the other the initially excited state. The latter appears as a zero-decay-time component whose amplitude can be readily quantified. The wavelength-dependent amplitude of this zero-lifetime component can be used to calculate the emission spectrum of the F state and. by difference, the emission spectrum of the relaxed state. For AcTrpNH₂ in propylene glycol at ?20°C the emission maxima of the F and R states are near 320 and 350 nm, respectively, and the relative proportion of the emission from each state was near 50%. At lower temperatures the emission from the F state becomes dominant and at high temperatures the emission from the R state dominates. We note that this resolution of states is somewhat arbitrary because we assumed a two-state model and the absence of solvent relaxed emission at 310 nm. Nonetheless, differential-wavelength deconvolution simplifies and facilitates the analysis of time-resolved fluorescence data from samples which undergo excited state reactions. Moreover, this deconvolution procedure considerably simplifies the determination of the kinetic constants for reversible excited state reactions. The application of differential-wavelength deconvolution does not increase the time reqaired for data acquisition. This differential analysis procedure should enhance the usefulness and precision of pulse fluorometric methods in studies of nanosecond time scale processes in proteins and membranes.     

Determination of symbiotic nodule occupancy in the model Vicia tetrasperma using a fluorescence scanner

Background

Fluorescent tagging of nodule bacteria forming symbioses with legume host plants represents a tool for vital tracking of bacteria inside the symbiotic root nodules and monitoring changes in gene activity. The constitutive expression of heterologous fluorescent proteins, such as green fluorescent protein (GFP), also allows screening for nodule occupancy by a particular strain. Imaging of the fluorescence signal on a macro-scale is associated with technical problems due to the robustness of nodule tissues and a high level of autofluorescence.

Scope

These limitations can be reduced by the use of a model species with a fine root system, such as Vicia tetrasperma. Further increases in the sensitivity and specificity of the detection and in image resolution can be attained by the use of a fluorescence scanner. Compared with the standard CCD-type cameras, the availability of a laser source of a specified excitation wavelength decreases non-specific autofluorescence while the photomultiplier tubes in emission detection significantly increase sensitivity. The large scanning area combined with a high resolution allow us to visualize individual nodules during the scan of whole root systems. Using a fluorescence scanner with excitation wavelength of 488 nm, a band-pass specific emission channel of 532 nm and a long-pass background channel of 555 nm, it was possible to distinguish nodules occupied by a rhizobial strain marked with one copy of cycle3 GFP from nodules colonized by the wild-type strain.

Conclusions

The main limitation of the current plant model and GFP with the wild-type emission peak at 409 nm is a sharp increase in root autofluorescence below 550 nm. The selectivity of the technique can be enhanced by the use of red-shifted fluorophores and the contrasting labelling of the variants, provided that the excitation (482 nm) and emission (737 nm) maxima corresponding to root chlorophyll are respected.     

Complex homogeneous and heterogeneous fluorescence anisotropy decays: enhancing analysis accuracy

In biological macromolecules, fluorophores often exhibit multiple depolarizing motions that require multiple lifetimes and rotational relaxation times to define fluorescence intensity and anisotropy decays. The related analysis of time-correlated single-photon counting data becomes uncertain due to the multitude of decay parameters and numerical sensitivity to deconvolution of the instrument response function (IRF) via discretization of integrals. By using simulations we show that improved discretizations based on quadratic and cubic local approximations of the IRF yield more accurate estimation of short rotational relaxation times and lifetimes than the commonly used Grinvald-Steinberg discretization, which in turn appears more reliable than two discretizations based on linear local approximations of the IRF. In addition, our simulation suggests that cubic approximation is the most advantageous in discriminating complex heterogeneous and homogeneous anisotropy decay. We show that among three different information criteria, the Akaike information criterion is best suited for detection of heterogeneity in rotational relaxation times. It is capable of detecting heterogeneity even when anisotropy decay appears homogeneous within statistical errors of estimation.     

Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes.

Nanosecond decays of the fluorescence anisotropy, r, were studied for the emission of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes. The relative contributions of the fast and the infinitely slow decaying component to the steady-state value r, of the fluorescence anisotropy were very similar for artifical and biological membranes. Angles, theta, of the cone, by which the motion of the fluorescent molecule is limited, were calculated from the intensity of the infinitely slow decaying anisotropy component and compared with steady-state fluorescence anisotropies and with 'microviscosities', (eta). An increase in (eta) from 1.5 to 5.2 P in our systems was accompanied by a decrease in theta from 49 degrees to 30 degrees while the decrease in the mean motional relaxation times, phi f, of the label molecule was not more than 1 ns and due mainly to changes in the potential, by which the diffusion of DPH in the membrane is restricted. From these observations we conclude that differences in the steady-state fluorescence anisotropy and in 'microviscosities' of cholesterol-containing membranes (r greater than 0.15) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.     

Interpretation of fluorescence correlation spectra of biopolymer solutions

Fluorescence correlation spectroscopy (FCS) is regularly used to study diffusion in non‐dilute “crowded” biopolymer solutions, including the interior of living cells. For fluorophores in dilute solution, the relationship between the FCS spectrum G(t) and the diffusion coefficient D is well‐established. However, the dilute‐solution relationship between G(t) and D has sometimes been used to interpret FCS spectra of fluorophores in non‐dilute solutions. Unfortunately, the relationship used to interpret FCS spectra in dilute solutions relies on an assumption that is not always correct in non‐dilute solutions. This paper obtains the correct form for interpreting FCS spectra of non‐dilute solutions, writing G(t) in terms of the statistical properties of the fluorophore motions. Approaches for applying this form are discussed. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 260–266, 2016.     

On the choice of laser dyes for use in exciting tyrosine fluorescence decays

The choice of laser dyes for exciting tyrosine fluorescence using synchronously pumped cavity-dumped dye laser systems is discussed. Rhodamine 560 was found to be optimal for a system based on an argon-ion pumping laser, whereas rhodamine 575 was preferred using a frequency-doubled Nd:YAG laser. Modifications of our fluorescence decay instrument to permit rejection of multiphoton events using a microchannel plate photomultiplier are described. An example of a four-component resolution of tyrosine decays illustrates the dramatic resolution capabilities attainable.     

Picosecond resolution of tyrosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry

We extended the technique of frequency-domain fluorometry to an upper frequency limit of 2000 MHz. This was accomplished by using the harmonic content of a laser pulse train (3.76 MHz, 5 ps) from a synchronously pumped and cavity-dumped dye laser. We used a microchannel plate photomultiplier as the detector to obtain the 2-GHz bandwidth. This new instrument was used to examine tyrosine intensity and anisotropy decays from peptides and proteins. These initial data sets demonstrate that triply exponential tyrosine intensity decays are easily recoverable, even if the mean decay time is less than 1 ns. Importantly, the extended frequency range provides good resolution of rapid and/or multiexponential tyrosine anisotropy decays. Correlation times as short as 15 ps have been recovered for indole, with an uncertainty of +/- 3 ps. We recovered a doubly exponential anisotropy decay of oxytoxin (29 and 454 ps), which probably reflects torsional motions of the phenol ring and overall rotational diffusion, respectively. Also, a 40-ps component was found in the anisotropy decay of bovine pancreatic trypsin inhibitor, which may be due to rapid torsional motions of the tyrosine residues and/or energy transfer among these residues. The rapid component has an amplitude of 0.05, which is about 16% of the total anisotropy. The availability of 2-GHz frequency-domain data extends the measurable time scale for fluorescence to overlap with that of molecular dynamics calculations.     

Resolution of fluorescence intensity decays of the two tryptophan residues in glutamine-binding protein from Escherichia coli using single tryptophan mutants.

Time correlated single photon counting measurements of tryptophan (Trp) fluorescence intensity decay and other spectroscopic studies were performed on glutamine-binding protein (GlnBP) from Escherichia coli. Using site-specifically mutated forms of the protein in which tyrosine (Tyr) and phenylalanine (Phe) substitute for the Trp residues at positions 32 and 220, we have examined whether wild-type (Wtyp) intensity decay components may be assigned to specific Trp residues. Results indicate that: (a) two exponential intensity decay components are recovered from the Wtyp protein (6.16 ns, 0.46 ns); (b) the long decay component arises from Trp-220 and comprises greater than 90% of the total fluorescence emission; (c) the short component arises from Trp-32 and is highly quenched; (d) all four single-Trp mutants exhibit multiexponential intensity decays, yet equimolar mixtures of two single-Trp mutants yield only two decay components which are virtually indistinguishable from the Wtyp protein; (e) the recovery of additional components in protein mixtures is obscured by statistical noise inherent in the technique of photon counting; (f) various spectroscopic measurements suggest that Trp-Trp interactions occur in the Wtyp protein, but the Wtyp intensity decay may be closely approximated by a linear combination of intensity decays from single-Trp mutants; and (g) inferences derived independently from fluorescence and NMR spectroscopy which pertain to the presence of Trp-Trp interactions and the relative solvent exposure of the two Trp residues are in agreement.     

A two-photon excitation fluorescence cross-correlation assay for a model ligand-receptor binding system using quantum dots

Two-photon excitation fluorescence cross-correlation spectroscopy (TPE-XCS) is a very suitable method for studying interactions of two distinctly labeled fluorescent molecules. As such, it lends itself nicely to the study of ligand-receptor interactions. By labeling the ligand with one color of fluorescent dye and the receptor with another, it is possible to directly monitor ligand binding rather than inferring binding by monitoring downstream effects. One challenge of the TPE-XCS approach is that of separating the signal due to the receptor from that of the ligand. Using standard organic fluorescent labels there is almost inevitably spectral cross talk between the detection channels, which must be accounted for in TPE-XCS data analysis. However, using quantum dots as labels for both ligand and receptor this limitation can be alleviated, because of the dot's narrower emission spectra. Using solely quantum dots as fluorescent labels is a novel approach to TPE-XCS, which may be generalizable to many pairs of interacting biomolecules after the proof of principle and the assessment of limitations presented here. Moreover, it is essential that relevant pharmacological parameters such as the equilibrium dissociation constant, K(d), can be easily extracted from the XCS data with minimal processing. Herein, we present a modified expression for fractional occupancy based on the auto- and cross-correlation decays obtained from a well-defined ligand-receptor system. Nanocrystalline semiconductor quantum dots functionalized with biotin (lambda(em) = 605 nm) and streptavidin (lambda(em) = 525 nm) were used for which an average K(d) value of 0.30 +/- 0.04 x 10(-9) M was obtained (cf. native system approximately 10(-15)). Additionally, the off-rate coefficient (k(off)) for dissociation of the two quantum dots was determined as 5 x 10(-5) s(-1). This off-rate is slightly larger than for native biotin-streptavidin (5 x 10(-6) s(-1)); the bulky nature of the quantum dots and restricted motion/orientation of functionalized dots in solution can account for differences in the streptavidin-biotin mediated dot-dot binding compared with those for native streptavidin-biotin.     

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.