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J R Lakowicz

Publications and source records attributed to J R Lakowicz.

At least 163 records · Page 9Linked to original sources
PubMed1988

Detection of three rotational correlation times for a rigid asymmetric molecule using frequency-domain fluorometry.

We measured the frequency response of the polarized emission of Yt-base in propylene glycol at 10 degrees C. Data were obtained for excitation wavelengths of 290, 312 and 346 nm, for which the fundamental anisotropies are 0.05, 0.19 and 0.32, respectively. Additionally, data were obtained using CCl4, to decrease the mean decay time from 9.1 to 4.2 ns. These nine sets of data were analyzed globally to recover the anisotropy decay law. Three correlation times were needed to fit the data, 0.8, 3.0 and 5.6 ns, a range of only 7-fold. We believe this is the first reported detection of three correlation times for a rigid molecule.

Guanine↗
PubMed1988

Conformation heterogeneity in proteins as an origin of heterogeneous fluorescence decays, illustrated by native and denatured ribonuclease T1.

We examined the frequency-domain intensity decays of the intrinsic tryptophan fluorescence (Trp-59) from ribonuclease T1 (EC 3.1.27.3) (RNAase T1). At pH 5.5 in the native state (below 30 degrees C), the intensity decay of the single tryptophan residue is a single-exponential process. Conditions which result in protein unfolding were found to induce more complex intensity decays. At temperatures above 40 degrees C, or in the presence of guanidine hydrochloride, the intensity decays became obviously double exponential. In general, the main effect of temperature or guanidine was to induce a second subnanosecond component in the intensity decay. The increased complexity of the decays could not be explained by a unimodal distribution of decay times. These results indicate that conformational dispersion of protein structure can be one origin of the multi-exponential decays which are generally observed for protein fluorescence.

Exoribonucleases↗
PubMed1988

Resolution of the lifetimes and correlation times of the intrinsic tryptophan fluorescence of human hemoglobin solutions using 2 GHz frequency-domain fluorometry.

We used 2 GHz harmonic content frequency-domain fluorescence to measure the intensity and the anisotropy decays from the intrinsic tryptophan fluorescence from human hemoglobin (Hb). The tryptophan intensity decays are dominated by a short-lived component which accounts for 35-60% of the total steady state intensity. The decay time of this short component varies from 9 to 27 ps and this component is sensitive to the ligation state of Hb. Our error analyses indicate the uncertainty is about +/- 3 ps. The intensity decays also show two longer lived components near 0.7 and 8 ns, which are probably due either to impurities or to Hb molecules in conformations which do not permit energy transfer. The anisotropy decays indicate the tryptophan residues in Hb are highly mobile, with apparent correlation times near 55 ps.

Carboxyhemoglobin↗
PubMed1988

Acrylamide quenching of Yt-base fluorescence in aqueous solution.

Acrylamide was found to be an effective quencher of Yt-base (Y-4,9-dihydro-4,6-dimethyl-9-oxo-1H-imidazo-1,2a-purine) in water. In the absence of collisional quenching the decay of Yt-base in water is predominantly a single exponential. The intensity decays become increasingly heterogeneous when quenched by acrylamide. The frequency-domain data were analyzed using the radiation model, which provides estimates of molecular parameters characteristic of the system. The mutual diffusion coefficient at 20 degrees C was found to be 0.5 x 10(5) cm2/s, the Yt-base acrylamide interaction radius was 8 A, and the rate constant for quenching was 100 cm/s. These values indicate that quenching is diffusion-limited, i.e., the encounter complex is deactivated at least 2-fold faster than the rate of diffusive encounters.

Acrylamide↗
PubMed1988

Frequency-domain measurements of the rotational dynamics of the tyrosine groups of calmodulin.

We used frequency-domain fluorometry to determine the intensity and anisotropy decay kinetics of tyrosine residues in calmodulin and its fragments. Excitation was provided by a continuous ultraviolet laser source, a frequency-doubled rhodamine 6G ring dye laser, whose output was externally modulated to 200 MHz. Both the intensity and anisotropy decays were found to be multiexponential and dependent upon temperature and solution conditions. By examination of calmodulin fragments we determined that energy transfer between the two tyrosine residues reduces the steady-state anisotropy values by about 20%. Additionally, the frequency-domain anisotropy decays indicate local torsional motions of the tyrosine residues, as well as significant individual motions of the two domains of calmodulin.

Calmodulin↗
PubMed1988

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.

Cyclohexanes↗
PubMed1988

Time-resolved emission spectra of hemoglobin on the picosecond time scale.

We used front-face illumination to examine the steady-state and time-resolved emission from the intrinsic tryptophan emission of human hemoglobin (Hb). Experimental conditions were identified which eliminated all contributions of scattered light. The sensitivity obtained using front-face optics was adequate to allow measurement of the wavelength-dependent frequency response of the emission to 2 GHz. The intensity decays displayed pico- and nanosecond components in the emission at all wavelengths from 315 to 380 nm. The contribution of the picosecond component decreased from 72 to 37% over this range of wavelengths. Frequency-domain measurements were used to calculate the time-resolved emission spectra and decay-associated emission spectra. These spectra indicate that the picosecond components of the emission display maxima near 320 nm, whereas the nanosecond components are centered at longer wavelengths near 335 nm. The nanosecond components appear to be due to residual impurities which remain even in highly purified samples of Hb. However, we cannot eliminate the possibility that some of these components are due to Hb itself.

Hemoglobins↗
PubMed1988

Gigahertz frequency-domain fluorometry: resolution of complex decays, picosecond processes and future developments.

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.

Lasers↗
PubMed1988

Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.

Energy Transfer↗
PubMed1987

Diffusion coefficients of quenchers in proteins from transient effects in the intensity decays.

We used 2-GHz frequency-domain fluorometry to examine the intensity decays of N-acetyl-L-tryptophamide (NATA) and the protein staphylococcal nuclease in the presence and absence of quenching by oxygen or acrylamide. When analyzed with a multiexponential model, the decays of NATA and nuclease both become more heterogeneous in the presence of quenching. We attribute the increased complexity to transient effects in quenching or equivalently a time-dependent rate constant for quenching. The frequency-domain data were analyzed using the Smoluchowski model (exp(-t/tau-2b square root t)) and the radiation model, which is known to correct some flaws in the more approximate Smoluchowski model. The radiation model provides improved fits to the data, as evidenced by average 10-fold decreases in chi R2. The radiation model also provides an estimate of the sum of the diffusion coefficients and the specific rate constant for quenching. The apparent diffusion coefficients for acrylamide and oxygen in nuclease, as seen by its single tryptophan (residue 140) are 15- and 11-fold lower than in water, respectively. The apparent values of the oxygen diffusion coefficient in water, as seen by NATA, are 2- to 3-fold larger than expected from earlier steady-state measurements. The ability to recover the detailed form of the intensity decays by the frequency-domain method should allow comparison of experimental results with calculated trajectories of quenchers in proteins.

Acrylamide↗
PubMed1987

Correction for contaminant fluorescence in frequency-domain fluorometry.

We describe a general method to correct for contaminant fluorescence when using the technique of frequency-domain fluorometry. The method can be applied regardless of the origin of the background signal, from scattered light, impurity fluorescence, or both. The procedure requires measurement of the frequency-dependent phase and modulation of the background at enough frequencies to approximate the decay law of the background. We also describe a general method to propagate the uncertainties in the measured phase and modulation values into the corrected values. This propagation is necessary to ensure proper weighting of the frequency-dependent data in the least-squares fitting algorithms. The practical usefulness of this correction method is demonstrated using frequency-domain data for one and two component mixtures which were deliberately contaminated with scattered light and/or other fluorophores.

Anthracenes↗
PubMed1987

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.

Kinetics↗
PubMed1987

Anisotropy decay of diphenylhexatriene in melittin-phospholipid complexes by multifrequency phase-modulation fluorometry.

Frequency-domain fluorometry was used to investigate the intensity and anisotropy decays of diphenylhexatriene (DPH) in melittin-lipid complexes. Simulated and experimental data indicate that correlation times ranging from 0.3 to 500 ns can be determined using data from 1 to 200 MHz. For the melittin-lipid complexes the hindered rotator model was not adequate to account for the anisotropy decays, especially at temperatures above the transition temperatures. At high protein-to-lipid ratios the data revealed the formation of small particles (100 A) of melittin and dipalmitoylphosphatidylcholine and the disruption of membrane order in bilayers of dipalmitoylphosphatidic acid.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1987

Intensity and anisotropy decays of the Wye base of yeast tRNA(Phe) as measured by frequency-domain fluorometry.

The intensity and anisotropy decays of Wye base fluorescence from yeast tRNA(Phe) were determined by frequency-domain fluorometry. The intensity decay is at least a double exponential in the presence and absence of Mg2+, but the multi-exponential character of the decay is more pronounced in the absence of Mg2+. The anisotropy decay displays components due to overall tRNA rotational diffusion and to local torsional motions. The amplitude of the local motion is decreased 2-fold by the presence of Mg2+. The results are broadly consistent with a more homogeneous environment for the Wye base in the presence of Mg2+.

Chelating Agents↗