Luminescent terbium protein labels for time-resolved microscopy and screening

Abstract

Brilliance of terbium: Heterodimeric conjugates of trimethoprim covalently linked to sensitized terbium chelates bind to Escherichia coli dihydrofolate reductase fusion proteins with nanomolar affinity (see picture). Terbium luminescence enables sensitive and time-resolved detection of labeled proteins in vitro and on the surface of living mammalian cells.

Figure 1

Intramololecular, time-resolved, fluorescence resonance energy transfer (TR-FRET) between eDHFR-bound TMP-TCs and GFP. Increasing concentrations of purified eDHFR-GFP were titrated against a constant concentration (20 nM) of each compound. Sensitized GFP emission (520 nm) was detected after a time delay of 100 μs, upon pulsed excitation with near-UV light (ca. 340 nm): green diamonds, TMP-Lumi4; red circles, TMP-cTTHA; blue x’s, TMP-cDTPA. Addition of 1 μM TMP reduced the signal, confirming FRET (shown for TMP-cTTHA, black squares). Lines represent non-linear least squares fit to the data.

Figure 2

Time-resolved microscopy of NIH3T3 cells treated with TMP-TCs. a) Overlay of bright field and prompt fluorescence (λ_ex = 480± 20 nm, λ_em = 535 ± 25 nm) images of cells transiently expressing nucleus-localized CFP and plasma membrane-localized eDHFR. b) Inverse, time-resolved fluorescence image of cells in a) showing non-specific luminescence. Cells were incubated 20 h in media containing TMP-cTTHA (100 μM), washed with PBS, mounted in media without compound, and imaged in time-resolved mode (λ_ex = 350± 25 nm, λ_em = 550± 10 nm, delay = 80 μs, exposure time = 1420 μs, no. exposure cycles = 660). c) Overlay image of cells transiently expressing nucleus-localized CFP and cell surface-localized eDHFR. d) Inverse, time-resolved fluorescence image of cells in c) showing membrane luminescence in transfected cell. Cells were incubated in media containing 1 μM TMP-Lumi4 (10 min.), washed, and imaged as in b).

Scheme 1

Conjugates of trimethoprim (TMP) to sensitised terbium chelate complexes. Top: cs124-polyaminocarboxylates (n=1, diethylenetriamine pentaacetic acid (TMP-cDTPA); n=2, triethylenetetraamine hexaacetic acid (TMP-cTTHA)). Bottom: Lumi4^®-Tb, a proprietary 2-hydroxyisophthalamide terbium complex (TMP-Lumi4).