Structural and Mechanistic Characterization of the Mannitol Transporter from E. coli using 5-fluorotryptophan as a Spectroscopic Probe

The mannitol permease (EIImtl) of E. coli is an integral membrane protein re- sponsible for the active transport of mannitol over the cytoplasmic membrane. It is composed of three domains: two cytosolic domains A and B, and trans- membrane C domain. The structures of A an B domains were solved by X- ray crystallography and NMR spectroscopy. For the transmembrane C domain a 5A ̊ 2D projection map is available and several topology models. EIImtl is functional as a dimer.
A dozen single Trp mutants of EIImtl were made and 5-fluoroTrp was in- corporated in the C domain with R 95% efficiency. Compared to Trp, 5- fluoroTrp shows the advantage that the fluorescence decay kinetics is much more homogeneous. 5-fluoroTrp is also a good energy donor, which makes it suitable for resonance energy transfer (RET) experiments. An an- alogue of mtl, azi-mtl, was used as an acceptor. Steady state fluorescence spectroscopy was used to characterize the solvent exposure of specific po- sitions within the transmembrane C domain. Time resolved fluorescence spectroscopy was used to probe the local microenvironment of the residues as well as the distance between 5-fluoroTrp residues and the mannitol binding site.
Our results show that mannitol binding induces large conformational changes in EIImtl, that the C domain shows a rigid structure and that the binding site is asymmetrically positioned in the EIImtl dimer.