Fluorescence Spectroscopy

3.4.1. Acquisition of Fluorescence Spectra

1. To avoid polarization artifacts fluorescence spectra must be taken under magic angle conditions. Therefore, place the excitation polarizer in vertical orientation (0°) and the emission polarizer at 54.7°.

2. Use a slit width for excitation and emission of approx 5 nm.

3. Correct for lamp fluctuation during acquisition by using 3 mg/mL Rhodamine B in glycerol as a quantum counter.

4. Dilute the labeled dsDNA to approx 100-150 nM and transfer 200-300 |L into a microcuvet (see Note 3).

5. Take FRET spectrum and acceptor spectrum with the following settings for the FRET pair fluorescein-TMRh:

FRET spectrum: Ex.: 490 nm Em.: 500-650 nm Acceptor spectrum: Ex.: 560 nm Em.: 570-650 nm The step size is 1 nm each.

6. Determine anisotropies of the donor and acceptor dye (see Subheading 3.4.2.). Keep the excitation polarizer in vertical position (0°) and measure the fluorescence intensity with the emission polarizer in vertical (0°, IVV) or horizontal orientation (90°, IVH) (see Note 4).

7. To measure the conformation of protein-DNA complexes add stepwise purified protein to the dsDNA solution until the FRET spectrum is unchanged. After each step wait approx 5-15 min for a complete binding of the protein to the dsDNA and repeat steps 5-7 (see Note 5).

8. Repeat the experiments with single-labeled dsDNA samples as controls. In the control experiments the fluorescence intensity of both dyes must remain constant.

3.4.2. Anisotropy

The anisotropy is an important parameter to translate the FRET efficiency into distance information. To assume an equal probability of all dipole orientations at least one dye should be able to rotate freely. The anisotropy is a measure for the rotational freedom of fluorescence dyes and values below 0.1 indicate a free rotation whereas values more than 0.2-0.25 indicate a limited rotation.

1. Excitation polarizer in vertical position (0°).

2. Measure fluorescence intensities with vertical (0°, IVV) and horizontal (90°, IVH) emission polarizer using following wavelength for excitation and emission (see Note 4):

Fluorescein: Ex.: 490 nm Em.: 518 nm TMRh: Ex.: 560 nm Em.: 590 nm

3. The anisotropy is given by:

r = (Iw - G ■ Ivh)/(Ivv + 2G ■ IVH) with G = IHV/IHH

4. For each experiment the instrumentation factor G needs to be determined only once for each dye (G is wavelength dependent). Therefore repeat step 2 with the excitation polarizer in horizontal position (90°).

3.4.3. Calculating the FRET Efficiency and Dye-to-Dye Distance

The intensity of the acceptor emission increases in the presence of FRET (sensitized emission) and the intensity of the donor emission correspondingly decreases. The spectral dispersions of the fluorescence intensities of the emission spectrum (FRET spectra) is fitted to the weighted sum of two spectral components: (1) a standard spectrum of the donor-only sample (donor spectrum), and (2) the fluorescence spectrum of the double-labeled sample (acceptor spectrum) excited at A,a, where only the acceptor absorbs (see Fig. 3).

FDD = a ■ FDD + (ratio)A ■ FDA a and (ratio)A are the fitted weighting factors of the two spectral components. The following protocol is for fluorescein as the donor and TMRh as the acceptor. For other dye-pairs the wavelength must be adjusted accordingly.

1. Fit the FRET spectrum FDD with the donor spectrum FDD from the donor-only sample over 500-540 nm; in this region only the donor fluorescein emits.

2. Subtract the fitted donor spectrum FDD from the FRET spectrum FDA resulting in the extracted acceptor spectrum FDD- aFDD.

3. (ratio)A is the extracted acceptor fluorescence signal normalized by FDA and can be determined by fitting the acceptor spectrum FDA to the extracted acceptor spectrum

Faa_ aFhA

DA D

4. (ratio)A is linearly dependent on the energy transfer efficiency E and normalizes the measured sensitized FRET signal for any errors in percentage of labeling.

(ratio)A = E ■ d+ ■ (edD/eaa) + (ea^) Calculate the FRET efficiency as follows:

E = {(ratio)a - (eaD/eaA)}/{d+ ■ (edD/eaa)} with (ea^) = (em^m*) = 0.095

d+ is the fraction of donor labeled molecules determined from the absorption spectrum (see Subheading 3.3.2.).

5. If at least one of the dyes can rotate freely as indicated by a low anisotropy (see Subheading 3.4.2. and Note 4) the measured FRET efficiency can be translated into an absolute dye-to-dye distance. The Forster distance R0 can be calculated with an average dipole orientation factor k2 of 2/3.

R = (1/E - 1)1/6 ■ R0(2/3) R0(2/3) is 50 A for fluorescein-TMRh.

Fig. 3. Fluorescence resonance energy transfer (FRET) analysis by sensitized emission. The FRET spectrum FDD (solid line) is fitted in the marked regions to (1) a standard donor spectrum FDD (dashed line) and (2) the acceptor fluorescence spectrum of the FRET sample FDA (dotted line). The subscripts DA and D indicate the FRET sample labeled with donor and acceptor molecules or the donor-only sample while the superscript indicates the excitation wavelength; and XA are the donor or acceptor excitation maxima, respectively (fluorescein 490 nm; 5-tetramethylrhodamine 560 nm). The ratio of the extracted acceptor spectrum FDD - aFDD normalized to the acceptor spectrum FDA, called (ratio)A, is linear dependent on the FRET efficiency E.

Fig. 3. Fluorescence resonance energy transfer (FRET) analysis by sensitized emission. The FRET spectrum FDD (solid line) is fitted in the marked regions to (1) a standard donor spectrum FDD (dashed line) and (2) the acceptor fluorescence spectrum of the FRET sample FDA (dotted line). The subscripts DA and D indicate the FRET sample labeled with donor and acceptor molecules or the donor-only sample while the superscript indicates the excitation wavelength; and XA are the donor or acceptor excitation maxima, respectively (fluorescein 490 nm; 5-tetramethylrhodamine 560 nm). The ratio of the extracted acceptor spectrum FDD - aFDD normalized to the acceptor spectrum FDA, called (ratio)A, is linear dependent on the FRET efficiency E.

4. Notes

1. To dry or equilibrate the Sephadex column with buffer place the 3-mL syringe in a 15-mL plastic tube and use a centrifuge holder for this tube size; the waste is collected in this tube. To collect the sample put a 1.5-mL Eppendorf tube and the column loaded with the nucleotide or dsDNA on top into a 50-mL plastic tube and centrifugate.

2. No air bubbles must be in the V-channel. Because of the electrical resistance of air no current conduction occurs and the DNA remains in the polyacrylamide gel.

3. A fluorophore concentration of 100-150 nM is easily detectable with fluorescence-spectrometer, is close to physiological levels and reduces the amount of protein necessary for the analysis. Higher fluorophore concentrations (>10-100 |M) can complicate the FRET analysis because of self-quenching. To decrease the sample volume furthermore small plastic or metal plates can be placed under the cuvet reducing the dead volume; the light beam passes the cuvet usually several millimeters above the bottom.

4. To determine the anisotropy no spectra need to be taken. Measure the fluorescence intensity five times with the emission polarizer in vertical and horizontal position and calculate the average for IVV and IVH for each dye. Typical anisotropy values for fluorescein and TMRh covalently bound at the DNA helical ends are 0.05-0.07 and 0.2-0.25, respectively. In protein-DNA complexes, these values are slightly higher owing to an increase of the complex size resulting in a reduced rotation capability of the whole complex. Furthermore, owing to an energy transfer and, therefore, a shorter fluorescence lifetime of the donor, the anisotropy of fluorescein is slightly higher in FRET samples than in a donor-only sample.

5. To avoid a dilution of the dsDNA solution in the cuvet and, thus, a decrease of the fluorescence signal after adding protein use concentrated protein solutions (10-20 ||M) if possible. If the dilution is not negligible, correct for the concentration decrease to compare, e.g., the donor fluorescence intensities of the free dsDNA and of the complex directly.

0 0

Post a comment