title: 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 a methodology for implementing this technique has been limited. We report here the design, expression, and characterization of a test protein for development of a LRET methodology. The protein, which we call DAL, contains the following domains (from the N-terminus): Escherichia coli dihydrofolate reductase (DHFR), the third and fourth ankyrin repeats of p16(INK4a), a lanthanide-binding tag (LBT), and a hexahistidine tag. LBT binds Tb(3+) with a submicromolar dissociation constant. LRET was measured from the Tb(3+) 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 p16(INK4a) are stably folded when combined with other protein domains. We found that Tb(3+) 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 Tb(3+) to Cu(2+) in the cytoplasm of live E. coli cells. The rates of Tb(3+) and Cu(2+) 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 effect on transport. Changing the external pH from 7 to 5 strongly inhibited the Tb(3+) signal, suggesting that the Tb(3+)-LBT interaction is useful as a cytoplasmic pH indicator in the range of approximately pH 5-6.
authors: Department of Biology, University of Texas at San Antonio, San Antonio, Texas 78249, United States.
authors: Daniel
authors: D
authors: González
authors: Nayanish
authors: N
authors: Lokhande
authors: Swaraj
authors: S
authors: Vadde
authors: Qi
authors: Q
authors: Zhao
authors: Aaron
authors: A
authors: Cassill
authors: Robert
authors: R
authors: Renthal
mesh_terms: D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D019793:Fluorescein / Q000378:metabolism; D049449:Luminescence; D008958:Models, Molecular; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D019793:Fluorescein