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

Abstract

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.

FIGURE 1:

Frequency-domain intensity decays for phenol, tyrosine, and rose bengal, 20°C. In each case, the symbols show the data and the solid lines the best single-exponential fits. (Top) Phenol (●); rose bengal (○). (Bottom) Tyrosine, pH 6.0 (●); tyrosine, pH 2.2 (○). The phase angle increases and the modulation decreases with increasing modulation frequency. See Table I for more details.

FIGURE 2:

Frequency-domain intensity decays of N-acetyl-l-tyrosinamide (top) and [Leu⁵] enkephalin (bottom) at 20°C. In each case, the symbols (●) represent the data, the solid line the best three-exponential fit, and the dashed line the best one-exponential fit. The phase angles increase and the modulations decrease with increasing modulation frequency. The decay and goodness of fit parameters are summarized in Table II.

FIGURE 3:

Frequency-domain intensity decays of oxytocin (top) and BPTI (bottom) at 25°C. The symbols (●) represent the data, the dashed line the best one-exponential fit, and the solid line the best three-exponential fit.

FIGURE 4:

Simulated frequency-domain data for rotational diffusion and segmental motions. The simulated data are for a intensity decay time of 2 ns, a rotational correlation time of 4 ns, and a segmental motion of 0.04 ns. The fundamental anisotropy (r₀) is assumed to be 0.30. The fractional amplitude of the 40-ps motion is 0.0 (- - -), 0.5 (—), and 1.0(⋯).

FIGURE 5:

Differential hase and modulated anisotropy data for NAcTyrA (▲) and [Leu⁵] enkephalin (○) at 20°C The NAcTyrA sample also contained 0.5 M KI.

FIGURE 6:

Differential phase and modulated data for oxytocin (●) and BPTI (▲) at 25°C.

FIGURE 7:

Picosecond anisotropy decays of N-acetyl-l-tyrosinamide in water. This sample contained 0.25 M KI to decrease the tyrosine decay time. The intensity decay parameters are in Table VI, and the anisotropy decay parameters are in Table IV. 5°C (▲); 58°C (●).

FIGURE 8:

Picosecond anisotropy decays of indole in methanol/water (75/25). The sample contained 0.25 M KI to decrease the decay time. The decay parameters are in Table V. 20°C (▲); 40°(●).

Scheme I