This paper presents a deceptively straightforward experimental approach to monitoring membrane protein-ligand interactions in vesicles and in living Escherichia coli cells. This is achieved via the biosynthetic incorporation of 7-azatryptophan, a tryptophan analogue with a red-shifted absorption spectrum, allowing collection of the emission signal of the target protein in a high tryptophan background via red-edge excitation. The approach is demonstrated for the mannitol permease of E. coli (EII mtl ), an integral membrane protein of 637 amino acids, including four tryptophans, and single-tryptophan mutants of EII mtl . By using a tryptophan auxotroph, a high level of 7-azatryptophan incorporation in EII mtl was achieved. The change in emission signal of the purified enzyme upon mannitol binding (-28%) was 4-fold larger than with EII mtl containing tryptophan, demonstrating the known higher sensitivity of this analogue for changes in the microenvironment [Schlesinger, R. (1968) J. Biol. Chem. 243, 3877-3883]. Changes in emission signal could also be monitored (-5%) when the enzyme was situated in vesicles, although it constituted only 10-15% of the total cytoplasmic membrane fraction. Of the five singletryptophan mutants, the emission signal of the mutant with 7-azatryptophan at position 198 was the most sensitive for mannitol binding. Changes in emission signal not only were observed in vesicles (-18%) but also could be monitored in viable cells (-5%). The fact that only modest expression levels and no protein purification are needed makes this a useful approach for the characterization of numerous protein systems under in vitro and in vivo conditions.Tryptophan fluorescence spectroscopy is a potentially powerful approach to investigating membrane proteins (1-4). The easy introduction of this probe at the DNA level and the sensitivity of tryptophan (Trp) 1 to changes in microenvironment facilitate the generation of structural and dynamic information at high resolution even at positions which are not solvent-exposed and, therefore, cannot be studied by the cysteine labeling approach (5, 6). Widespread use of Trp fluorescence spectroscopy in membrane protein research has been hampered however by the difficulty of eliminating high background fluorescence, a prerequisite for quenching and especially for time-resolved lifetime and anisotropy measurements. Recently, we have addressed this issue and developed procedures for avoiding high background fluorescence (4,7,8).Membrane vesicles are an ideal system for investigating membrane protein-ligand interactions, yet the cysteine labeling and the Trp spectroscopy methodologies usually necessitate the solubilization and purification of the labeled protein, followed eventually by its reconstitution back in proteoliposomes. Besides being laborious, the purification often affects the functional and oligomeric state of the membrane protein and its interactions with other proteins in the case of multiprotein complexes. Moreover, the difficulty of unidirectional recon...