Wieslaw Wiczk

A 10‐GHz frequency‐domain fluorometer

We developed a frequency‐domain fluorometer which operates from 4 to 2000 MHz. The modulated excitation is provided by the harmonic content of a laser pulse train (3.76 MHz, 5 ps) from a synchronously pumped and cavity dumped dye laser. The phase angle and modulation of the emission are measured with a microchannel‐plate photomultiplier (PMT). Cross‐correlation detection is performed outside the PMT. The high‐frequency signals for cross correlation were obtained by multiplication of the output from a 500‐MHz frequency synthesizer. The performance was verified in several ways, including measurement of known time delays and examination of standard fluorophores. The detector displayed no detectable color effect, with the 300–600‐nm difference being less than 5 ps. The precision of the measurements is adequate to detect differences of 20 ps for decay times of 500 ps. A correlation time of 53 ps was found for indole in water at 20u2009°C. The shortest correlation time we measured was 15 ps for indole in methanol/w...

Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer.

We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.

Resolution of a distribution of distances by fluorescence energy transfer and frequency-domain fluorometry

We used frequency-domain fluorescence spectroscopy to examine the intensity decays of a fluorescent donor attached to an acceptor via a flexible alkyl chain. The intensity decay of the indole donor becomes markedly heterogeneous due to energy transfer to the dansyl acceptor. The measured dispersion of fluorescence decay times was used to recover the donor-to-acceptor distance distribution. The distance distribution was found to be characteristic of the molecule, and not the model used for data analysis. The ability to recover distance distributions in solution should be valuable in studies of biological macromolecules.

Lifetime distributions and anisotropy decays of indole fluorescence in cyclohexane/ethanol mixtures by frequency-domain fluorometry

We used frequency-domain fluorometry to measure intensity and anisotropy decay of indole fluorescence in cyclohexane/ethanol mixtures at 20 degrees C. In 100% cyclohexane or 100% ethanol the intensity decay of indole appears to be a single exponential with decay times of 7.66 and 4.10 ns, respectively. In cyclohexane containing a small percentage of ethanol (up to 10%), we observed increased heterogeneity in intensity decay, resulting in a 10-fold increase in chi 2R for the single-exponential fit, as compared with the double-exponential model. We obtained comparable or better fits using unimodal Lorentzian and Gaussian lifetime distributions (two floating parameters) than for the two-exponential model (three floating parameters). We believe that the distribution of decay times reflects a range of indole solvation states in the dominately nonpolar solutions. This result suggests that a variety of hydrogen-bonding configurations could be one origin of the distributions of decay times observed for tryptophan emission from proteins. We also measured rotational diffusion of indole in cyclohexane, ethanol and its mixtures at 20 degrees C. The picosecond correlation times required that the mean decay times be decreased by acrylamide quenching (in ethanol) or energy transfer (in cyclohexane). In ethanol we observed nearly isotropic rotation of indole; in cyclohexane we obtained two correlation times of 17 and 73 ps. The shorter correlation time in cyclohexane appears to be due to the slip boundary condition, which was found to be progressively eliminated by small percentages of ethanol. Hence, hydrogen-bonding interactions appear to have a substantial effect on the rotational dynamics of indole.

End-to-end distance distributions of flexible molecules from steady state fluorescence energy transfer and quenching-induced changes in the Förster distance

Abstract We describe a new method to recover the distribution of distances between two sites on flexible molecules, which requires only steady state measurements of fluorescence intensities. We used collisional quenching of the donor fluorescence to vary the characteristic Forster distance ( R 0 ) for energy transfer. The measured transfer efficiencies for each value of R 0 provide different samples of the distance distribution. The R 0 -dependent transfer efficiencies, when analyzed by non-linear least squares, were found to reliably determine the distance distribution, as shown by evaluation of the uncertainties in the distribution and by agreement with the frequency-domain method (Chem. Phys. Letters 138 (1987) 587).

Intramolecular dynamics in the environment of the single tryptophan residue in staphylococcal nuclease.

The dipole relaxational dynamics in the environment of a single tryptophan residue Trp-140 in staphylococcal nuclease was studied by time-resolved (multi-frequency phase-modulation) spectroscopy and selective red-edge excitation. The long-wavelength position of the fluorescence spectrum (at 343 nm) and the absence of red-edge excitation effects at 0 and 20 degrees C indicate that this residue is surrounded by very mobile protein groups which relax on the subnanosecond time scale. For these temperatures (0-20 degrees C) the steady-state emission spectra did not show the excitation-wavelength dependent shifts (red-edge effects) for excitation wavelengths from 295 to 308 nm; however, the anisotropy decay rate is slow (tens of nanoseconds). This suggests that the spectral relaxation is due to mobility of the surrounding groups rather than the motion of the tryptophan itself. The motions of the tryptophan surrounding are substantially retarded at reduced temperatures in viscous solvent (60% glycerol). The temperature dependence of the difference in position of fluorescence spectra at excitation wavelengths 295 and 305 nm demonstrate the existence of red-edge effect at sub-zero temperatures, reaching a maximum value at -50 degrees C, where the steady-state emission spectrum is shifted to 332 nm. The excitation and emission wavelength dependence of multi-frequency phase-modulation data at the half-transition point (-40 degrees C) demonstrates the existence of the nanosecond dipolar relaxations. At -40 degrees C the time-dependent spectral shift is close to monoexponential with the relaxation time of 1.4 ns.

Site-to-site diffusion in proteins as observed by energy transfer and frequency-domain fluorometry.

Abstract We report measurements of the site‐to‐site diffusion coefficients in proteins and model compounds, which were measured using time‐dependent energy transfer and frequency‐domain fluorometry. The possibility of measuring these diffusion coefficients were shown from simulations, which demonstrate that donor (D)‐to‐acceptor (A) diffusion alters the donor frequency response, and that this effect is observable in the presence of a distribution of donor‐to‐acceptor distances. For decay times typical of tryptophan fluorescence, the simulations indicate that D‐A diffusion coefficients can be measured ranging from −7 to −5 cm2/s. This possibility was verified by studies of a methylenechain linked D‐A pair in solutions of varying viscosity. The D‐A diffusion was also measured for two labeled peptides and two proteins, melittin and troponin I. In most cases we used global analysis of data sets obtained with varying amounts of collisional quenchers to vary the donor decay time. Unfolding of troponin I results in more rapid D‐A diffusion, whereas for melittin more rapid diffusion was observed in the α‐helical state but over a limited range of distances.

New trends in photobiology: Gigahertz frequency-domain fluorometry: Resolution of complex decays, picosecond processes and future developments

Abstract We describe the principles, instrumentation and applications of frequency-domain fluorescence spectroscopy. This method is useful for the resolution of multi-exponential decays and complex anisotropy decays on the picosecond timescale. The present instrumentation allows measurements to 2 GHz, which has been used to measure rotational correlation times as short as 7 ps. In the future it may be possible to extend the frequency range to 10 GHz, which should allow still faster processes to be quantified. It should be emphasized that resolution of fast processes is not obtained at the expense of losing information on the nanosecond timescale. Additionally, the GHz frequency-domain measurements are performed using low excitation intensities, which do not damage the samples.

Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1: Distance distributions by frequency-domain fluorometry

The separation between Cys 697 (SH1) and Cys 707 (SH2) of the heavy chain of myosin subfragment-1 was previously measured by fluorescence resonance energy transfer with a donor linked to SH1 and an acceptor to SH2. In the present study the distribution of the distances between the two thiols was recovered from frequency-domain fluorometry. In the native state and in the presence of ligands such as MgADP, pyrophosphate, orthovanadate (Vi) and actin, we found wide distributions of the separations between SH1 and SH2 (11-16 A) comparable to that found in the random-coil state (20 A). These results suggest that the SH1-SH2 segment has a high degree of conformational flexibility even in native S1. The flexibility is not much affected by the physiological state of S1. However, the ligands MgADP, Vi and MgADP + Vi decrease significantly the mean SH1-SH2 distance from 27 to 17 A with the effect of MgADP+ Vi being the most pronounced. The anisotropy decay of donor-labeled S1 is biphasic with two rotational correlation times. The long component is decreased by these ligands from 289 to 93 ns, suggesting a more compact symmetric structure of S1 in the presence of the ligands. The complex S1(MgADP)Vi has been shown to be a stable analogue of S1(MgADP)Pi, an unstable intermediate that is generated in the actomyosin ATPase cycle during muscle contraction. Since the power stroke of muscle is accompanied by release of Pi from S1(MgADP)Pi, the present results are consistent with a model in which force generation can be accompanied by transition of S1 from a highly symmetric or compact structure to a more extended structure.

Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry

We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.