Figure 1.
Photochemical and biochemical properties of the purified mTagBFP2 protein.
(A) Time dependence of fluorescence for mTagBFP2, mTagBFP and EBFP2 in PBS, pH 7.4 at 37°C. (B) Excitation (dashed line), absorbance and emission (solid lines) spectra of mTagBFP2. (C) Photobleaching curves for purified mTagBFP2 (squares) and mTagBFP (circles) under epifluorescence illumination using metal halide arc lamp. According to the Student's t-test [21] a difference between the photobleaching curves is statistically significant. (D) Dependence of fluorescence half-life times on pH for mTagBFP2 and mTagBFP at 37°C. (E) Time dependence of fluorescence for mTagBFP2 and mTagBFP denatured in 6 M guanidinium hydrochloride at 25°C. Error bars, s.d. (n = 3 (A, D, E); and n = 10 (C)).
Table 1.
Properties of purified mTagBFP2 in comparison with other monomeric blue fluorescent proteins.
Figure 2.
Autocatalytic degradation of the TagBFP-like chromophore.
(A) Absorbance spectra of mTagBFP2 (blue) and mTagBFP (black) before and after incubation in 100 mM NaH2PO4, 300 mM NaCl, pH 8.0 at 37°C for 200 h. (B) Time dependence of absorbance for mTagBFP2 (blue) and mTagBFP (black) at 330 nm (dashed lines) and 401 nm (solid lines). Purified proteins were incubated in 100 mM NaH2PO4, 300 mM NaCl, pH 9.0 at 37°C. (C) SDS-PAGE analysis of mTagBFP2 and mTagBFP after incubation at 4°C (lanes 2 and 3) or at 37°C (lanes 4 and 5) for 770 h. M: molecular weight protein markers. Bands 1 and 2 show the total polypeptide chain and the polypeptide chain after cleavage inside the chromophore, respectively. (D) The ratio of the band 1 intensity to the band 2 intensity for SDS-PAGE analysis presented in Figure 2C. (E) Deconvoluted FT-ICR mass spectrum: isotopic distribution of the region corresponding to the band 2 (C). The deconvoluted average mass (19163.83 Da) is indicated by the red arrow. (F) Chemical scheme of hydrolysis of the mTagBFP-like chromophore. Error bars, s.d. (n = 2 (B); and n = 3 (D)).
Figure 3.
Properties of mTagBFP2 in live mammalian cells.
Photobleaching curves for mTagBFP2 (squares) and mTagBFP (circles) expressed in HeLa cells under metal halide arc illumination (A) or 405 nm laser scanning confocal microscope illumination (B). Data represent an average of 32–65 cells per each protein. According to the Student's t-test [21] a difference between the photobleaching curves is statistically significant. (C) Mean fluorescence brightness of HeLa cells expressing mTagBFP2 (solid line) or mTagBFP (dashed line). (D) Mean fluorescence of mTagBFP2- and mTagBFP-expressing HeLa cells corresponding to panel (C). Error bars, s.d. (n = 65 (A); n = 32 (B); and n = 3 (C)).
Figure 4.
Fluorescence imaging of mTagBFP2 fusion constructs.
(A–M) C-terminal fusion constructs. For each fusion protein, the linker amino acid length is indicated after the name of the targeted organelle or fusion protein. (A) mTagBFP2-lamin B1-10; (B) mTagBFP2-CAAX-farnesyl-5; (C) mTagBFP2-endoplasmic reticulum-5 (calreticulin and KDEL); (D) mTagBFP2-fibrillarin-7; (E) mTagBFP2-light chain clathrin-15; (F) mTagBFP2-β-actin-7; (G) mTagBFP2-caveolin 1-10; (H) mTagBFP2-vinculin-22; (I) mTagBFP2-CAF1-10 (chromatin assembly factor); (J) mTagBFP2-Rab5a-7; (K) mTagBFP2-α-tubulin-18; (L) mTagBFP2-myosin-IIA-18; (M) mTagBFP2-PCNA-19. (N–X) N-terminal fusion constructs. (N) Cx26-mTagBFP2-7; (O) TfR-mTagBFP2-20 (transferrin receptor); (P) Golgi-mTagBFP2-7; (Q) zyxin-mTagBFP2-6;(R) VE cadherin-mTagBFP2-10; (S) mitochondria-mTagBFP2-7; (T) CENPB-mTagBFP2-22; (U) α-actinin-mTagBFP2-19; (V) c-src-mTagBFP2-7; (W) Lifeact-mTagBFP2-7; (X) vimentin-mTagBFP2-7. The cell line used for expressing mTagBFP2 fusion vectors was opossum kidney cortex proximal tubule epithelial cells (ATCC CRL-1840) in panel X, and human cervical adenocarcinoma cells (HeLa; ATCC CCL-2) in the remaining panels. The scale bar in each panel equals 10 µm.
Figure 5.
Behavior of mTagBFP and mTagBFP2 as FRET donors.
(A) mTagBFP-mEGFP; (B) mTagBFP-mEmerald; (C) mTagBFP2-mEGFP; (D) mTagBFP2-mEmerald. Left column: emission spectra of FRET pairs with excitation at 405 nm before (black lines) and after (red lines) acceptor photobleaching. Columns 2 and 3: fixed cells in blue (Ex. 405 nm, Em. 445–470 nm) and green (Ex. 488 nm, Em. 500–535 nm) fluorescence channels after acceptor photobleaching in the square region (black square in the green channel). Column 4: FRET efficiency in fixed cells. Columns 5–6: live cells in blue/green channel before (column 5) and after (column 6) acceptor photobleaching are shown. Column 7: FRET efficiency in live cells. Colored scale bars (columns 4 and 7) show relative FRET efficiencies. See also Table 2.
Table 2.
Analysis of FRET efficiencies in live and fixed HeLa cells.