Ben H. Hesp

Vibrational Relaxation in Neat Crystals of Naphthalene by Picosecond CARS

Abstract Picosecond delayed CARS experiments on totally symmetric modes in naphthalene at 1.5 K are reported. The Raman lineshape of the vibrational excitons is lorentzian and vibrational relaxation can be surprisingly slow. The Raman lineshape of the A g exciton level of the 766 cm −1 vibrational mode reveals that the low-temperature lorentzian lineshape occurs by motional narrowing At higher temperature the exciton is trapped by interaction with lattice phonons.

Localization of the Substrate-binding Site in the Homodimeric Mannitol Transporter, EIImtl, of Escherichia coli

The mannitol transporter from Escherichia 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, 19 single Trp-containing mutants of EIImtl were biosynthetically labeled with 5-fluorotryptophan (5-FTrp) and mixed with azi-mannitol, a substrate analog acting as a Förster resonance energy transfer (FRET) acceptor. Typically, for mutants showing FRET, only one 5-FTrp was involved, whereas the 5-FTrp from the other monomer was too distant. This proves that the mannitol-binding site is asymmetrically positioned in dimeric IICmtl. Combined with the available two-dimensional projection maps of IICmtl, it is concluded that a second resting binding site is present in this transporter. Active transport of mannitol only takes place when EIImtl becomes phosphorylated at Cys384 in the cytoplasmic B domain. Stably phosphorylated EIImtl mutants were constructed, and FRET experiments showed that the position of mannitol in IICmtl remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol.

Emitting State of 5‑Hydroxyindole, 5‑Hydroxytryptophan, and 5‑Hydroxytryptophan Incorporated in Proteins

5-Hydroxy-L-tryptophan (5HW) has been biosynthetically incorporated in many proteins to facilitate their characterization using fluorescence spectroscopy. An attractive feature of this tryptophan analogue is its absorbance at 310-320 nm, allowing its specific excitation in a Trp background. The red-shift in absorbance upon introduction of a hydroxyl group at the 5-position of Trp or indole was found to be due to a lowering of the (1)Lb transition energy. It was therefore believed that 5HW only features (1)Lb emission. Recently, calculations for 5-hydroxyindole (5HI) in water revealed (1)La is the emitting state, and the same was predicted for 5HW incorporated in proteins. To clarify which state emits in 5HI and 5HW, we present here excitation anisotropy spectra of these probes and of four proteins labeled with 5HW at a surface exposed position. Our data clearly show (1)Lb is the emitting state of 5HI, 5HW, and 5HW in three of the proteins investigated. For one protein mixed emission was observed, and the decay kinetics were found strongly dependent on the emission wavelength. This work provides the first experimental evidence that (1)La can be the emitting state for this Trp analogue incorporated in a protein.

Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy.

The mannitol transporter EII(mtl) from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EII(mtl) is functional as a dimer and its membrane-embedded C domain, IIC(mtl), harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IIC(mtl). At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IIC(mtl). Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EII(mtl) is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IIC(mtl) structure are involved in the sugar translocation. The structure of EII(mtl), as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EII(mtl), the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.

Structure of the Cytoplasmic Loop between Putative Helices II and III of the Mannitol Permease of Escherichia coli: A Tryptophan and 5-Fluorotryptophan Spectroscopy Study †

In this work, four single tryptophan (Trp) mutants of the dimeric mannitol transporter of Escherichia coli, EII(mtl), are characterized using Trp and 5-fluoroTrp (5-FTrp) fluorescence spectroscopy. The four positions, 97, 114, 126, and 133, are located in a region shown by recent studies to be involved in the mannitol translocation process. To spectroscopically distinguish between the Trp positions in each subunit of dimeric EII(mtl), 5-FTrp was biosynthetically incorporated because of its much simpler photophysics compared to those of Trp. The steady-state and time-resolved fluorescence methodologies used point out that all four positions are in structured environments, both in the absence and in the presence of a saturating concentration of mannitol. The fluorescence decay of all 5-FTrp-containing mutants was highly homogeneous, suggesting similar microenvironments for both probes per dimer. However, Stern-Volmer quenching experiments using potassium iodide indicate different solvent accessibilities for the two probes at positions 97 and 133. A 5 A two-dimensional (2D) projection map of the membrane-embedded IIC(mtl) dimer showing 2-fold symmetry is available. The results of this work are in better agreement with a 7 A projection map from a single 2D crystal on which no symmetry was imposed.

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.