5-Fluorotryptophan as Fluorescent Probe to Characterize an Oligomeric Membrane Protein

Abstract

The mannitol transporter from E. coli, EIImtl, belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EIImtl is functional as a homodimer and it harbors one high-affinity mannitol binding site in the membrane-embedded C domain (IICmtl). To localize this binding site, single Trp containing mutants of EIImtl were mixed with azi-mannitol, a substrate analogue acting as a Förster resonance energy transfer (FRET) acceptor (R0 of 10 Å). Due to the complex fluorescence decay of Trp, we could not establish whether one or both Trp residues showed FRET with azi-mannitol. To overcome this, we took advantage of the homogeneous decay of 5-fluorotryptophan and this analog was biosynthetically incorporated in 19 mutants. Typically, for mutants showing FRET, only one 5-FTrp was involved, while the 5-FTrp from the other monomer was too distant. This proves that the mannitol binding site is asymmetrically positioned in dimeric IICmtl. The FRET results localized the position of the binding site halfway the first transmembrane helix. Combined with available 2D projection maps of IICmtl, it is concluded that a second resting binding site is present in this transporter. This work demonstrates the potential in structural and mechanistic protein research of a donor-acceptor pair with a very short R0, of which the donor shows homogeneous fluorescence decay kinetics.