Luminescence Resonance Energy Transfer in the Cytoplasm of Live Escherichia coli Cells

Abstract

Luminescence resonance energy transfer (LRET) offers many advantages for accurate measurements of distances between specific sites in living cells, but progress in developing methodology for implementing this technique has been limited. We report here the design, expression, and characterization of a test protein for development of LRET methodology. The protein, which we call DAL, contains the following domains (from the N-terminus): E. coli dihydrofolate reductase (DHFR), the third and fourth ankyrin repeats of p16INK4a, a lanthanide-binding tag (LBT), and a hexahistidine tag. LBT binds Tb3+ with a sub-micromolar dissociation constant. LRET was measured from the Tb3+ site on LBT to transition metals bound to the hexa-His tag, and to fluorescein methotrexate bound to DHFR. The measured distances were consistent with a molecular model constructed from the known crystal structures of the constituent domains of DAL. The results indicate that the two C-terminal ankyrin domains of p16INK4a are stably folded when combined with other protein domains. We found that Tb3+ binds to DAL in the cytoplasm of live E. coli cells, and thus DAL is useful as an indicator for studies of metal transport. We also used DAL to measure LRET from Tb3+ to Cu2+ in the cytoplasm of live E. coli cells. The rates of Tb3+ and Cu2+ transport were not affected by a proton uncoupler or an ATP synthase inhibitor. Reversal of the membrane potential had a small inhibitory effect, and removal of lipopolysaccharide had a small accelerating affect on transport. Changing the external pH from 7 to 5 strongly inhibited the Tb3+ signal, suggesting that the Tb3+-LBT interaction is useful as a cytoplasmic pH indicator in the range of about pH 5-6.