Figure 1.
Illustration of the FRET assay.
(a) A schematic diagram illustrating the phenomena of FRET with Alexa Fluor 488 conjugated LFA-1 as the donor and Alexa Fluor 555 conjugated D1-D2-Fc as the acceptor. When there is no binding between LFA-1 and D1-D2-Fc, excitation of donor at 470 nm leads to 520 emission peak only. However, when there is binding between LFA-1 and D1-D2-Fc, excitation at 470 nm leads to acceptor emission at 570 nm, due to FRET. (b) Normalized absorbance and emission spectra of Alexa Fluor 488 and Alexa Fluor 555. (c) UV- visible absorption spectra for fluorophore-protein conjugates (for the determination of the molecular concentration of fluorophores and proteins).
Figure 2.
Ascertaining the FRET activity between Alexa Fluor 488-LFA-1 and Alexa Fluor 555-D1-D2-Fc.
Fluorescence emission spectra of individual fluorophore-protein conjugates, Alexa Fluor 488-LFA-1 (100 nM) and Alexa Fluor 555-D1-D2-Fc (100 nM), and a mixture of Alexa Fluor 488-LFA-1 (100 nM) and Alexa Fluor 555-D1-D2-Fc (100 nM) are compared. A sensitized acceptor emission at 570 nm and a donor quenching at 520 nm are observed in the emission spectra of the FRET mixture, confirming FRET. For control, the emission spectrum of the mixture of the dyes (Alexa Fluor 488+ Alexa Fluor 555), 100 nM each is also shown. That fact that no emission peak appeared at 570 nm for Alexa Fluor 555 when only the dye mixture (Alexa Fluor 488+ Alexa Fluor 488) was excited at 470 nm, whereas a prominent acceptor sensitized peak was observed for the FRET mixture (Alexa Fluor 488-LFA-1+ Alexa Fluor 555-D1-D2-Fc) indicates that the random FRET between the free dye molecules can be neglected in our study. All the spectra were obtained under the excitation of 470 nm. The gain of the spectrofluorometer was set at 100 (manual). The excitation and the emission bandwidths were fixed at 9 nm (for 316–850 nm excitation range) and 20 nm (for 280–850 nm emission range) for all the measurements. The fluorescence emissions were recorded with an integration time of 20 µs (more details on Table S1).
Figure 3.
Determination of the FRET emission signal at 570 nm.
(a) Fluorescence emission spectra (when excited at 470 nm) of Alexa Fluor 488-LFA-1 and Alexa Fluor 555-D1-D2-Fc mixtures, wherein the Alexa Fluor 488-LFA-1 concentration was fixed at 100 nM and that of Alexa Fluor 555- D1-D2-Fc was varied from 0 to 1.6 µM; (b) Emission signal of the donor (FD) in the mixture at 520 nm when excited at 470 nm; (c) Emission signal of Alexa Fluor 555 at 570 nm (FA) in the mixture when excited at 530 nm; (d) Total emission signal of the mixture at 570 nm when excited at 470 nm. From (b), we see that the quenching of the donor is negligible when the acceptor concentration exceeds ∼500 nM, which indicates that the increased concentrations of acceptor gradually saturate the number of donor binding pairs.
Figure 4.
Determination of the ratio constants, a and b.
(a) To obtain a, fluorescence emission spectra of Alexa Fluor 488-LFA-1 alone at three different concentrations (100, 200, and 300 nM), excited with 470 nm, were obtained. To calculate b, fluorescence emission spectra of Alexa Fluor 555-D1-D2-Fc only at several concentrations (100, 200, 300, 400, and 500 nM) upon excitation at 530 nm (b) and 470 nm (c) were obtained.
Figure 5.
Determination of dissociation constant (Kd) and maximum FRET emissions (FFRETmax) signal.
The plot shows the fitting curve of FRET emission signal with equation (6). The FFRETmax and the corresponding Kd of the LFA-1/D1-D2-Fc interaction were determined to be 6.33×103 RFU and 17.93±1.34 nM, respectively. The flattening of the FRET emission signal, when the acceptor concentration exceeds ∼500 nM, can be attributed to the fact that the increased acceptor concentration gradually saturates the number of donor binding pairs.
Figure 6.
Determination of A/D ratio for optimal FRET between Alexa Fluor 488-LFA-1 and Alexa Fluor 555-D1-D2-Fc.
Fluorescence emission scans were obtained for the Alexa Fluor 488-LFA-1 and Alexa Fluor 555-D1-D2-Fc mixture, where the concentration of Alexa Fluor 555-D1-D2-Fc was kept constant at 100 nM and that of Alexa Fluor 488-LFA-1 was varied: 25 nM (A/D = 8.40), 50 nM (A/D = 4.20), 100 nM (A/D = 2.12), 150 nM (A/D = 1.41), 200 nM (A/D = 1.06), 250 nM (A/D = 0.84) and 300 nM (A/D = 0.70). The mixtures were excited at 470 nm. The highest FRET efficiency (∼53.51%) was obtained for A/D = 2.12. For both 4.20 and 8.40 A/D ratios, the donor peak intensity is very small compared to the acceptor peak, while for 2.12 A/D ratio, the donor peak intensity is higher but not overwhelming the acceptor peak intensity. These higher emission counts at 4.20 and 8.40 A/D can be attributed to the direct emission of the acceptor as the acceptor concentration exceeds the saturating concentration required to saturate the donor binding pairs.
Table 1.
A/D Ratio and FRET Efficiency (%).
Figure 7.
A summary of the inhibitory effects (to the LFA-1/ICAM-1 interactions) of (a) lovastatin; and (b) LFA-1 derived peptides.
“*” and “**” denote statistical significance with p<0.05 and p<0.005, respectively, from student t-test. NS: Not Significant. In (a), different concentrations of lovastatin were added to the FRET mixture of Alexa Fluor 488- LFA-1 (100 nM) and Alexa Fluor 555-D1-D2-Fc (100 nM). The inhibition efficiency of lovastatin increased from 69.48±0.77% at 0.2 µM to 90.03±0.06% at 200 µM. This result confirms that this FRET based screening assay is capable of identifying/classifying inhibitors of the LFA-1/ICAM-1 interaction based on inhibition efficiency study. From (b), a comparison of the inhibition efficiencies (to the LFA-1/ICAM-1 interactions) of three LFA-1 derived peptides CD11a237–261, CD11a441–465, and CD11a456–465 indicates that CD11a237–261 exhibits the highest inhibition efficiency while CD11a456–465 the lowest for all the concentrations tested in this study.