The use of light quenching to selectively eliminate the emission of biochemical fluorophores based on the
emission wavelength is described. To demonstrate the possibility of wavelength-selective light quenching, a
mixture of two fluorophores, 4-(dimethylamino)-4'-cyanostillene (DCS) and Prodan, emitting at different
wavelengths was examined first. The emission spectrum and intensity decay were altered by the 570-nm quenching pulse due to selective quenching of the longer wavelength emission of DCS. Quenching of the
solvent-sensitive fluorophore partially bound to human serum albumin and partially in the aqueous phase
was then examined. Light quenching with a long wavelength (570 nm) time-delayed pulse selectively
quenched the Prodan fluorophore in the aqueous phase while in the presence of the Prodan bound to human
serum albumin, which emitted at shorter wavelengths, was not quenched. Using one-beam short wavelength
excitation and quenching, a selective quenching of the blue-shifted emission of ribonuclease T1 in the presence of the red-shifted emission of the tryptophan residue in adrenocorticotropic hormone was observed. In both systems wavelength-selective light quenching was demonstrated by a shift in the emission spectra, and by changes in the intensity decay consistent with preferential quenching of one species. Light quenching is instantly reversible by blocking or defocusing of the quenching beam. It can occur for inaccessible residues or in viscous solvents, and thus can be of wide applicability for resolving the complex emission of biological macromolecules.
Frequency-domain fluorometry was used to investigate the time- dependent intensity decays of N-acetyl-L-tryptophanamide (NATA) when collisionally quenched by acrylamide and iodide in propylene glycol at 20 degree(s)C. The intensity decays of NATA became increasingly heterogeneous in the presence of quenching, and the steady-state Stern-Volmer plots show significant upward curvature. These frequency-domain and steady-state data clearly indicate that the rate constants for quenching of NATA by acrylamide and iodide depend exponentially on the fluorophore- quencher separation distance. The NATA intensity decays were not consistent with the Smoluchowski-Collins-Kimball radiation boundary condition for quenching. The upward curvature of the Stern-Volmer plots provide a sensitive method to determine the characteristic distances for fluorophore-quencher interaction. The rate constant for quenching are discussed based on the mechanism of interaction between tryptophanyl fluorophore and quencher molecules which appear to involve electron transfer for acrylamide and heavy atom effects for iodide. These results suggest that the distance-dependent interaction and the effects of solvent dynamics need to be considered in the interpretations of data for quenching of proteins.
The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state of the art time-resolved fluorescence instrumentation and software for scientists, whose research can be enhanced by such experimental data. The CFS is a national center, supported by the National Center for Research Resources Division of the National Institutes of Health, and in part by the National Science Foundation. Both time-domain (TD) and frequency- domain (FD) measurements (10 MHz to 10 Ghz) are available, with a wide range of excitation and emission wavelengths (UV to NIR). The data can be used to recover distances and site-to-site diffusion in protein, interactions between macromolecules, accessibility of fluorophores to quenchers, and the dynamic properties of proteins, membranes and nucleic acids. Current software provides for analysis of multi-exponential intensity and anisotropy decays, lifetime distribution, distance distributions for independent observation of fluorescence donors and acceptors, transient effects in collisional quenching, phase-modulation spectra and time-resolved emission spectra. Most programs provide for global analysis of multiple data sets obtained under similar experimental conditions. Data can be analyzed on-site by connection with the CFS computers through the internet. During six years of operation we have established scientific collaborations with over 30 academic and industrial groups in the United States. These collaborations have resulted in 63 scientific papers.
Recent experimental results from this laboratory have shown that fluorescence emission can be quenched by the light pulses from cavity-dumped dye lasers, a phenomenon we call `light quenching.' In this overview article we describe some of the possible effects of light quenching on the steady state and time-resolved spectral properties of fluorophores. The extent of light quenching was found to depend on the amplitude of the emission spectrum at the quenching wavelength. Different effects are expected and were observed for light quenching by a single laser beam (within a single laser pulse) or for a time-delayed quenching pulse. Light quenching can decrease or increase the time-zero anisotropy. Our calculations indicate that the anisotropies can increase to unity under selected conditions. Remarkably, the light quenching can break the usual z-axis symmetry of the excited state population, and the measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. Quenching with time-delayed light pulses is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. A more complete description of `One and Two-Pulse Theory of Light Quenching,' is presented by Kusba et al. in this same volume. The predicted oscillations have been observed in frequency-domain intensity decay data. Overall, the results suggest a new class of two-pulse or multiple-pulse time-resolved experiments where the sample is prepared by the excitation pulse and subsequent modification of the excited state population by the quenching pulse(s), followed by time- or frequency-domain measurements of the resulting emission.
KEYWORDS: Diffusion, Energy transfer, Luminescence, Molecules, Resolution enhancement technologies, Molecular energy transfer, Proteins, Solids, Distance measurement, Fluorescence resonance energy transfer
We describe a method to improve the resolution of donor-to- acceptor distance distributions in molecules which are flexing on the timescale of the fluorescence lifetime. We measured the time- dependent donor decays of two donor (D)-acceptor (A) pairs, where the donor lifetimes were substantially different. The donors were an indole residue (5.7 ns) and a naphthalene residue (24.4 ns). The same dansyl acceptor was used for both D-A pairs. The donor decays are complex due to both a distribution of D-A distances and D-A diffusion. Using the donor decay data for each D-A pair alone, it is difficult to resolve both the distance distribution and the D-to-A diffusion coefficient. However, these values are unambiguously recovered from global analysis of the data from both D-A pairs. Simulations were also used to demonstrate the increased reduction of global analysis with different lifetime donors to obtain distance distribution parameters in the presence of D-A diffusion.
We have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high-repetition rate lasers, a phenomenon we call 'light quenching'. We now describe some of the possible effects of light quenching on the time- resolved intensity and anisotropy decays of fluorophores. We show that light quenching can decrease or increase the time-zero anisotropy, and that the initial anisotropies can increase to unity under selected conditions. Quenching with time-delayed light pulses is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. The increasing available and use of pulsed laser sources of light offers the opportunity for a new class of one- two- or multiple-pulse time- resolved experiments.
The effect of the collisional quenching on the fluorescence intensity decays has been studied by frequency-domain fluorometry. We used an efficient (CBr4) and/or inefficient (CCl4 quencher to quench the fluorescence of 1,2-benzanthracene (1,2-BA). The wide range of diffusion has been obtained by using propylene glycol at different temperatures (-40 degree(s)C to 40 degree(s)C). The measured intensity decays cannot be satisfactorily fitted either to the Smoluchowski or Collins-Kimball (RBC) model, except the case of inefficient quencher in the presence of high diffusion. In particular, we observed quenching in diffusionless conditions (-40 degree(s)C). To describe the collisional quenching of the fluorescence more correctly we propose a new model which includes a distance-dependent quenching rate (DDQ model). The DDQ simulations show that the local concentration of quencher surrounding the excited fluorophore cannot be approximated by using the RBC model, except in the case of high diffusion and low quenching rate. The DDQ model describes well all measured intensity decays of 1,2-benzanthracene in the presence of CBr4 and/or CCl4. Also, the DDQ model more correctly predicts the curvature of Stern-Volmer plots and activation energies obtained from the temperature dependent rate of quenching.
The energy transfer among several fluorophores when bound to linear DNA has been studied. The intercalation and groove binding of the fluorophores and relatively large persistent length of DNA makes it a good model for one dimensional energy transfer. In this case, as predicted by Foerster's theory, the donor intensity decayed with a t1/6 time dependence. The presence of a finite volume with a restricted geometry leads to significantly different donor intensity decays from that predicted by Foerster's model. We used intercalated/groove-bound fluorophores as donors and transition metal ion complexes which only bind on the outside surface of the DNA as acceptors, to characterize energy transfer in a cylindrical geometry. Two models were used: a hard cylinder with a donor on the z-axis and acceptors on the surface, and a soft-boundary cylinder where a distribution of acceptors within a cylindrical volume was allowed. The energy transfer among intercalated/groove-bound donors and surface-bound acceptors in DNA can be described with soft-boundary cylindrical geometry with reasonable parameters.
Measurements of time-resolved intramolecular energy transfer in progressively stretched poly(vinyl alcohol) films were performed. The donor (tryptophan) and acceptor (dansyl) were linked with flexible polymethylene chain. Distance distributions were recovered from frequency-domain measurements of the donor decay. In isotropic PVA (in solution) a wide range of distances were detected (Gaussian full width at half maximum of about 16 angstroms) with an average distance of 13 angstroms. The donor-acceptor distance distribution became progressively more narrow when the PVA films were stretched. Four-fold or more stretching results in a single donor-acceptor distance. The maximal measured donor-acceptor distance of 23 angstroms is in excellent agreement with the computed distance for the fully stretched conformation. The possibility of partial and/or full ordering of polymethylene chains by stretching the PVA films can also be useful in the study of other distance-dependent interactions, such as electron transfer or distance-dependent quenching.
We report measurements of site-to-site diffusion in proteins, using frequency-domain measurements of time-dependent energy transfer. The possibility of such measurements is shown from simulations which demonstrate that donor-to-acceptor (D-to-A) diffusion alters the donor frequency response, and that this effect is observable in the presence of a distribution of distances. For decay times typical of tryptophan fluorescence, the simulations indicate D-to-A diffusion coefficients can be measured ranging from 10-7 to 10-5 cm2/s. This possibility was verified by studies of a methylene-chain linked D-A pairs in solutions of varying viscosity. D-to-A diffusion was also measured for acceptor-labeled melittin in the random coil and (alpha) -helical states. Unfolding of troponin I results in increased D-A diffusion. Surprisingly, more rapid diffusion was observed for melittin in the (alpha) -helical state, but over a limited range of distances.
Measurements of time-resolved fluorescence are increasingly used for research in biophysics, biochemistry, cell biology and medicine. Advances in the technology of light sources and detectors are resulting in more reliable and/or advanced instrumentation, which is resulting in the expanding applications of fluorescence spectroscopy. Time-resolved measurements are often performed by direct measurements in the time-domain. In this article the authors describe the alternative method of frequency-domain fluorometry. The frequency-response of the emission to intensity-modulated excitation can be used to recover the time-dependent decay. Commercial instrumentation now allows measurements to an upper light modulation frequency limit of 200 MHz. This laboratory has developed second and third generation instruments which allows measurements to 2 GHz and subsequently to 10 GHz. The frequency-domain data from such instrumentation provides excellent resolution of picosecond decays of intensity and anisotropy. Additionally, the frequency-domain method appears to provide remarkable resolution of complex decays which are often observed for biochemical samples. In this article the authors describe this instrumentation and applications of this method. Examples are shown using probes with ps decay and correlation times, the intrinsic fluorescence of proteins, and the measurement of end-to-end diffusion in proteins and/or flexible molecules.
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