Structural studies of the melibiose permease of Escherichia coli by fluorescence resonance energy transfer. I. Evidence for ion-induced conformational change.

Further insight into the cosubstrate-induced structural change of the melibiose permease (MelB) of Escherichia coli has been sought by investigating the binding and spectroscopic properties of the fluorescent sugar 2'-(N-5-dimethylaminonaphthalene-1-sulfonyl)aminoethyl 1-thio-beta-D-galactopyranoside (Dns2-S-Gal) and related analogs (Dns3-S-Gal or Dns6-S-Gal with a propyl or hexyl instead of an ethyl linker, respectively) interacting with MelB in membrane vesicles or in proteoliposomes. The three analogs efficiently inhibit melibiose transport and bind to MelB in a sodium-dependent fashion. Their dissociation constants (Kd) are in the micromolar range in the presence of NaCl and an order of magnitude higher in its absence. In the presence of NaCl and Dns2-S-Gal, sample excitation at 335 or 297 nm gives rise to a fluorescent signal at around 465 nm, whereas Dns3-S-Gal or Dns6-S-Gal emits a fluorescence light at 490 or 506 nm, respectively. Detailed study of the Dns2-S-Gal signal elicited by a 297 nm illumination indicates that a tryptophan-mediated fluorescence resonance energy transfer phenomenon is involved in the response. All fluorescence signals below 500 nm are prevented by addition of melibiose in excess, and the kinetic constants describing their dependence on the probe or NaCl concentrations closely correlate with the probe binding constants. Finally, the Dns2-S-Gal signal recorded in sodium-free medium is red shifted by up to 25 nm from that recorded in the presence of NaCl. Taken together, these results suggest (i) that the fluorescence signals below 500 nm arise from Dns-S-Gal molecules bound to MelB, (ii) the presence of a highly hydrophobic environment close to or at the sugar-binding site, the polarity of which increases on moving away from the sugar-binding site, and (iii) that the interaction of sodium ions with MelB enhances the hydrophobicity of this environment. These results are consistent with the induction of a cooperative change of the structure of the sugar-binding site or of its immediate vicinity by the ions.

The melibiose permease (MelB) 1 of Escherichia coli is an integral membrane protein that couples the entry of several ␣-galactosides, such as melibiose or some ␤-galactosides, to either Na ϩ , Li ϩ , or H ϩ by a symport or cotransport mechanism (for reviews, see Ref. 1). MelB is encoded by the melB gene and is a member of the super family of homologous sodium/solute symporters (2,3). It consists of 473 amino acids (molecular mass, 53 kDa), 70% of which are apolar (4,5). Hydropathy predictions, gene fusion analysis, limited proteolysis, and immunological studies indicate that MelB is a polytopic protein with 12 membrane domains connected by alternated periplasmic or cytoplasmic loops of short or limited size (6 -8). A Histagged melibiose permease has been purified by affinity chromatography and shown to be solely responsible for symport activity in proteoliposomes (5).
Kinetic studies (9), mutagenesis experiments (10 -14), and studies of MelB chimeras (15) all suggest that Na ϩ , Li ϩ , and H ϩ share a common binding site buried, at least in part, in the N-terminal membrane domain of the transporter. This site contains several acidic and polar residues that may be involved in the coordination of either monovalent ions or H 3 O ϩ (rather than H ϩ ) (11). In addition, intrinsic fluorescence studies of a purified His-tagged transporter suggest that cosubstrate binding induces cooperative modifications in MelB conformation (16). Thus, whereas MelB fluorescence emission is slightly quenched by the monovalent ions or moderately enhanced by adding the sugar in a sodium-or lithium-free medium, addition of both substrates induces a large fluorescence change. Subsequent studies have shown that Trp 299 and Trp 342 , located on helices IX and X of MelB, respectively, contribute most of the ␣-galactoside-induced fluorescence signal increase (17). One or more tryptophans from the N-terminal domain are also involved in the fluorescence response on adding a ␤-galactoside. Interestingly, the polarity of Trp 299 or Trp 342 environment decreases upon sugar binding, suggesting that they become less accessible to the solvent. Altogether, these data suggest that the structure of the sugar-binding site, hypothetically located in the C-terminal domain, is directly modified by the sugar and is further modified in a cooperative and indirect fashion upon the binding of the coupling ion to the N-terminal domain.
Further insight into the cosubstrate-induced change of MelB structure can be obtained by probing more directly the sugarbinding site properties with sugar analogs carrying an appropriate reporter. Fluorescent galactosides, such as (N-dan-syl)aminoalkyl-1-thio-␤-D-galactopyranosides (Dns-S-Gal), seem particularly suited as the dansyl fluorescence properties depend on solvent polarity, providing a means to assess the physicochemical properties of the substrate binding site (or of its immediate proximity) and their variation during catalysis. Previous of the spectroscopic analyses of Dns-S-Gal derivative-Lac carrier interactions in E. coli membranes illustrate this approach (18 -21).
In the present study, we show that several Dns-S-Gal derivatives are high affinity ligands of MelB in native membranes or of purified MelB in proteoliposomes. The overlap between protein tryptophan fluorescence emission and Dns-S-Gal absorbance has been exploited to investigate specific aspects of the MelB-cosubstrate interaction using fluorescence resonance energy transfer (FRET) spectroscopy. The results provide information on the environment of Dns-S-Gal molecules bound to MelB. It is shown that the polarity of this environment is modified in a sodium-dependent fashion. Together with cosubstrate-induced intrinsic fluorescence variation data (16,17), these results are used to speculate on the structural properties of the sugar-binding site of MelB.
The dansyl galactosides Dns 2 -S-Gal, Dns 3 -S-Gal, and Dns 6 -S-Gal were kindly provided by Dr. H. R. Kaback (HHMI/UCLA), and Dns 0 -S-Gal was a gift from Dr. M. Page (Hoffman-LaRoche, Basel, Switzerland). The purity of the different fluorescent compounds was verified by mass spectra and NMR analyses. Stock solutions of the fluorescent derivatives (10 mM) were prepared in dimethyl sulfoxide.
Permease Activity-Right-side-out membrane vesicles were prepared using an osmotic shock procedure (24) and washed in 0.1 M potassium phosphate (pH 7). Transport activity was assessed by measuring the time course of [ 3 H]melibiose (20 mCi/mmol) accumulation using a filtration assay as described previously (25). Binding of ␣-[ 3 H]NPG (0.8 Ci/mmol) to RSO membrane vesicles or to proteoliposomes was assayed under nonenergized conditions in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (10 M) and monensin (0.75 M) using a flow dialysis procedure (9). All experiments were carried out at 20°C.
MelB Purification and Reconstitution in Liposomes-Freshly transformed cells were grown to an A 600 ϭ 2 at 30°C as described previously (16). Inverted membrane vesicles were prepared by means of a French press (American Instrument Co., 13,000 psi), and MelB purification was carried out as described by Pourcher et al. (5). MelB reconstitution was performed in the presence of a 5-fold excess of E. coli lipids and using detergent adsorption on Bio-Beads SM-2 (26). Proteoliposomes were submitted to repeated freezing/thawing-sonication-wash cycles in nominally Na ϩ -free, 10 mM potassium phosphate buffer (pH 7) to eliminate NaCl from the internal space. Flame photometry measurements indicated that the level of contaminating sodium salts in the proteoliposome solutions was below 20 M. Purity of the reconstituted MelB (generally Ͼ98%) was assessed from silver-stained SDS-polyacrylamide gel electrophoresis gels.
Fluorescence Measurements-Steady state fluorescence measurements were performed with a LS50B Perkin-Elmer spectrofluorometer equipped with 1-cm-path length cuvettes, subjected to magnetic stirring, and thermostatted at 20°C. Experiments were carried out on briefly sonicated proteoliposomes resuspended at 15 g of protein/ml in Na ϩ -free, 100 mM potassium phosphate buffer (pH 7) and preequilibrated at 20°C. Each emission fluorescence spectrum is the mean of three scans recorded at 0.5-nm step intervals and with an integration rate of 1 s/nm. All illustrated spectra are corrected for the baseline signal recorded in the absence of Dns-S-Gal. When experiments were carried out on sonicated liposomes, the liposome concentration was adjusted to give a final absorbance similar to that of the proteoliposome suspension (A 400 about 0.1). The specific spectral contribution of Dns 2 -S-Gal interacting with MelB ( em [max] at 465 nm) was corrected for nonspecific contribution using the signal recorded in a NaCl-free medium containing melibiose in excess (100 mM).
Protein Assays and Determination of Contaminating Sodium Salts-Proteins contained in membrane vesicles and proteoliposomes were assayed according to Ref. 27 using serum bovine albumin as a standard. The concentration of contaminating sodium salts in the sodium-free medium was measured by flame photometry. The Dns-S-Gal binding constants (K d ) were estimated by carrying a competition assay in the presence of ␣-[ 3 H]NPG and proteoliposomes carrying purified MelB (9). Table I shows that the K d for Dns 2 -S-Gal, Dns 3 -S-Gal, or Dns 6 -S-Gal is, in every case, in the micromolar range when NaCl is present at a concentration that fully activates sugar binding on MelB (10 mM). Comparatively, Dns 0 -S-Gal is a poor inhibitor as it reduces ␣-[ 3 H]NPG binding by less than 5% when present at a final concentration of 50 M (data not shown). Table I also shows that all K d values are systematically higher in the absence of activating sodium cations, establishing that Dns-S-Gal binding to MelB is Na ϩ -dependent. Overall, the results demonstrate that Dns-S-Gals are high affinity substrates of MelB.

Dansylated Galactosides
Fluorescence Properties of the DG Analogues Interacting with Membranes-Deenergized DW2-R/pK31⌬AHB membrane vesicles, incubated in a medium containing 10 mM NaCl and 15 M Dns 2 -S-Gal, were illuminated at two different wavelengths, and the fluorescence signal was recorded in the 400 -570-nm interval. ex was first set at 335 Ϯ 5 nm to excite the Dns 2 -S-Gal molecules directly ( Fig. 2A). The fluorescence emission was recorded before (upper trace) and after (lower trace) addition of melibiose in excess (40 mM). Both spectra exhibit a major peak at about 540 nm originating from free Dns 2 -S-Gal molecules in solution. It is significant that the shoulder observed in the interval 420 -490 nm before addition of melibiose (upper trace) disappears after its addition (lower trace). The difference spectrum (with or without melibiose), representing Dns 2 -S-Gal specifically bound to MelB, has a maximum at about 465 nm ( Fig.  2A, inset). Comparatively, addition of a poor substrate of MelB, such as sucrose, had no effect on the signal (not shown). Similar features occurred if the sample was excited at ex ϭ 297 Ϯ 5 nm (Fig. 2B). The 420 -520 nm fluorescent signal (upper trace) disappears on adding melibiose in excess (lower trace) and has, again, a maximum at about 465 nm (inset). Although not shown, no significant 420 -520 nm signal was recorded from RSO-membrane vesicles lacking MelB. Fig. 3 shows that the fluorescence emission of Dns 2 -S-Gal, Dns 3 -S-Gal, or Dns 6 -S-Gal specifically bound to MelB varies with analog. Following excitation at 297 nm (or at 335 nm, not shown), em [max] of the Dns 2 -S-Gal signal was about 465 nm, whereas those of Dns 3 -S-Gal and Dns 6 -S-Gal were 490 and 502 nm, respectively. All signals were only detected in the presence of Na ϩ ions in the assay medium. The Dns 3 -S-Gal and Dns 6 -S-Gal signals were not further characterized.
Evidence of a Fluorescence Resonance Energy Transfer Process between the Permease Trps and Bound Dns 2 -S-Gal in Proteoliposomes-The fluorescence signal recorded from MelB pro-teoliposomes was analyzed in the 310 -570 nm interval to assess the implication of permease Trps in the Dns 2 -S-Gal fluorescence signal upon illumination at 297 Ϯ 5 nm (Fig. 4A). In the absence of Dns 2 -S-Gal and NaCl, the fluorescence contribution in the 310 -400 nm interval is typical of the transporter Trps (Fig. 4A, spectrum a) (16). Addition of 15 M Dns 2 -S-Gal gave rise to a small signal between 420 and 540 nm and a peak at 540 nm typical of dansylated sugars in an aqueous medium (Fig. 4A, spectrum b). The Trps fluorescence signal also decreased, in part because of an inner filter effect and a nonspecific quenching of the Trp signal by Dns 2 -S-Gal from the medium (data not shown). Subsequent addition of 10 mM NaCl  induced not only a drastic increase in light emitted between 420 and 540 nm but at the same time a quenching of the Trps fluorescence signal (Fig. 4A, spectrum c). Finally, these spectral changes were reversed by adding melibiose in excess (100 mM) (Fig. 4A, spectrum d), i.e. the 420 -540 nm signal decreases, and at the same time, the Trps signal increased or even exceeded the level recorded before addition of NaCl. The calculated difference spectrum (spectra c-d) (i.e. Ϯ melibiose) has a maximum at 465 nm (Fig. 4A, inset). Fig. 4B shows that no such fluorescence signal changes were observed in liposome suspensions. Finally, it is worth mentioning that the sodium-dependent increase in fluorescence signal recorded at 465 nm (Ϯ5 nm) has an excitation spectrum with two distinct maxima (not shown). That at 335 nm is proportionally small, NaCl-insensitive, and overlaps the Dns 2 -S-Gal absorption spectrum. The other maximum (285 nm) is about 3 times greater, overlaps the Trps absorption area, is enhanced upon addition of NaCl, and decreases when melibiose is added. Taken together, these data establish that the sodium-induced rise of the fluorescence signal in response to an illumination at 297 nm ( em [max] at 465 nm) is at least largely due to a fluorescence resonance energy transfer phenomenon between the permease Trps and Dns 2 -S-Gal molecules specifically bound to the transporter. This signal is hereafter referred to as the (Trp 3 Dns)-FRET signal.
Characterization of the (Trp 3 Dns)-FRET Phenomenon-In MelB proteoliposomes incubated in the presence of a concentration of NaCl that fully activates MelB (10 mM), the specific (Trp 3 Dns)-FRET signal saturated as a function of Dns 2 -S-Gal (Fig. 5). Half maximal signal variation was reached at a Dns 2 -S-Gal concentration of 1.5 M (Fig. 5B). This value compares well with the Dns 2 -S-Gal K d value determined by binding studies (Table I). In addition, the concentration of NaCl that produces half maximal activation of the (Trp Acylation of MelB cysteine(s) by N-ethylmaleimide impairs the translocation but not the binding of MelB substrates (10). Exposing membrane vesicles or proteoliposomes to 0.5 mM of NEM for 30 min had no significant effect on the characteristics of the sodium-dependent (Trp 3 Dns)-FRET signal (not shown). These data indicate that the (Trp 3 Dns)-FRET phenomenon is associated with events occurring at the stage of substrate binding to the permease. Fig. 4 indicate that the (Trp 3 Dns)-FRET signal from MelB proteoliposomes is much more important in the presence of 10 mM NaCl than in its absence. To assess whether the sodium-induced signal variation is consecutive to a modification of the energy donor (MelB Trps) and/or acceptor (bound Dns 2 -S-Gal) properties, we analyzed the fluorescence signal elicited by an excitation at 335 or at 297 nm in media before and after addition of NaCl (Fig. 6). For this comparison, the fraction of permeases interacting with Dns 2 -S-Gal was kept constant by incubating the proteoliposomes in the presence of 1.5 M Dns 2 -S-Gal in NaCl-containing medium or 15 M in sodium-free medium, i.e. at analog concentration corresponding to the binding constant in each medium. At both excitation wavelengths (Fig. 6, A and B), the fluorescence signal amplitude was much higher in the sodiumcontaining (upper spectra) than in the sodium-free (lower spectra) solution. More important, the em

Sodium-induced Change in the Microenvironment of Dns 2 -S-Gal Bound to MelB -The results shown in
[max] signal in sodiumfree medium was about 490 or 475 nm upon direct or indirect excitation, respectively (Fig. 6, A and B, lower traces), whereas

FIG. 4. Evidence of a FRET phenomenon between MelB tryptophans and Dns 2 -S-Gal in proteoliposomes.
A, sonicated proteoliposomes harboring purified His 6 -tagged MelB (15 g) were equilibrated in 1 ml of nominally Na ϩ -free 10 mM potassium phosphate (pH 7) and the appropriate ionophores (see Fig. 1B) at 20°C. The sample was illuminated at 295 nm (Ϯ 5 nm), and the emission fluorescence was recorded between 310 and 570 nm before any addition (a) or after the consecutive additions of Dns 2 -S-Gal at 15 M (b), 10 mM NaCl (c, heavy line), and finally 100 mM melibiose added as dry powder (d, dotted line). Each spectrum is the mean of three scans. Inset, specific MelB bound Dns 2 -S-Gal FRET signal calculated from the difference between the spectra recorded before and after addition of melibiose. B, sonicated E. coli phospholipids (used to prepare the proteoliposomes) were incubated in Na ϩ -free 10 mM potassium phosphate (pH 7) and the appropriate ionophores. Their concentration was adjusted to give an absorbance at A 400 similar to that of the proteoliposome suspension used above. Spectra a and b were recorded before and after addition of Dns 2 -S-Gal at 15 M, respectively. it was 465 nm at both ex in the presence of NaCl (Fig. 6, A and  B, upper traces). In the nominally sodium-free medium used in our experiments, the fluorescence signal is made up of two components: one arising from the H ϩ /Dns 2 -S-Gal/carrier complexes and the other from a small but significant fraction of Na ϩ /Dns 2 -S-Gal/carrier complexes that are formed in the presence of the contaminating sodium salts (15 M). Considering that the sodium activating K 0.5 [act] is 0.05 mM(see above), one can estimate that the Na ϩ -dependent or H ϩ -dependent component in sodium-free medium represents 65 or 35% of the signal recorded during direct excitation (Fig. 7A) and 80 or 20% of the recorded FRET signal, respectively (Fig. 7B). In both instances, the deduced H ϩ -dependent component has a em [max] above 495 nm. These data indicate that the acceptor environment is more polar in the absence than in the presence of the coupling sodium ions. Moreover, the ratio of the FRET and direct fluorescence signals computed from the H ϩ -dependent component is at least half that calculated from the sodium-dependent signals. Keeping in mind that this ratio is dependent on the amount of light absorbed by the acceptor but independent of its environment (and therefore quantum yield), its value provides insight into the effect of sodium ions on the donor properties and/or donor-acceptor relationships. DISCUSSION The experiments reported in the present study show that additional insight into the cosubstrate-induced structural change of the MelB transporter of E. coli can be obtained by analyzing the spectral properties of different fluorescent 2Ј-(Ndansyl) aminoalkyl 1-thio-␤-D-galactopyranosides interacting with MelB in membrane vesicles or proteoliposomes. The results suggests that on interacting with MelB, the activating sodium ion modifies the hydrophobicity of the immediate environment of the transporter sugar-binding site, probably as a result of structural change occurring in domains close to or forming this sugar-binding site.
The dansylated sugars Dns 2 -S-Gal, Dns 3 -S-Gal, and Dns 6 -S-Gal fulfill all of the criteria required for being considered high affinity substrates of MelB. At the micromolar level, they in-hibited active transport of melibiose (Fig. 1A) or competed with the high affinity ligand ␣-NPG for binding on MelB in deenergized RSO membrane vesicles (Fig. 1B). In addition, they bound to MelB in a sodium-dependent fashion (Table I) and were displaced by melibiose in a competitive-like fashion. The affinity of MelB for the Dns-S-Gal analogs is 2 orders of magnitude higher than that of the physiological substrate melibiose (around 0.5 mM) but is comparable to that of ␣-NPG (0.6 M). In agreement with previous contention, the data suggest that although the specificity of the sugar-MelB interaction is primarily dictated by the galactosyl configuration, grafting an aromatic group on the glycosidic linkage strongly stabilizes the sugar-transporter interaction (see Ref. 1 and references therein). Overall, these data strongly support the contention that Dns-S-Gal analogs bind specifically to the sugar-binding site of MelB.
Two lines of evidence indicate that Dns-S-Gal molecules specifically bound to the transporter are responsible for the generation of the fluorescence signal with em [max] below 500 nm recorded on irradiating membrane vesicles or MelB proteoliposomes at either 335 or 297 nm. The first is kinetic and is illustrated by the close correlation existing between the spectral and binding properties of Dns 2 -S-Gal. Both processes are sodium-dependent, progressively titrated by increasing concentration of melibiose, and described quantitatively by using similar kinetic constants. As selective inactivation of the substrate translocation activity of MelB has no significant effect on sugar binding or on the fluorescence response means that the fluorescence signal monitors events occurring at the stage of substrate binding to the permease. The spectral characteristics of the sodium-dependent fluorescence signal also suggest a direct relationship between the spectroscopic and Dns-S-Gal binding to MelB. Thus, the em [max] value recorded in the presence of Dns 2 -S-Gal (465 nm) or even that of Dns 3 -S-Gal (490 nm), is far below that attributed to the same analog interacting nonspecifically with E. coli membranes or lipids (514 -520 nm) (21). Also, one would not expect much change in the emission maximum of different sugar analogs nor a strong dependence on the presence of NaCl if the fluorescent signal were to arise from dansyl galactosides partitioned into the membrane lipid core or not specifically interacting with MelB. Incidentally, it should be noted that the em [max] of the fluorescence signal emitted by Dns 2 -S-Gal bound to E. coli Lac permease is around 493 nm rather than 465 nm as for MelB (18,21), indicating that the signal is specific to the transporter studied. Finally, the concomitant and reciprocal variation of Trp and Dns 2 -S-Gal fluorescence signals in proteoliposomes illuminated at 297 nm and the appearance of the 280-nm component in the dansylated sugar excitation spectrum support the conclusion that the fluorescence signal recorded at 465 nm results from a fluorescence resonance energy transfer phenomenon ((Trp 3 Dns)-FRET)) between the tryptophan residues of MelB and the probe. Emission of Dns 2 -S-Gal at about 465 nm in the presence of NaCl suggests that the dansyl reporter is located in a highly hydrophobic microenvironment with an apparent dielectric constant equivalent to that of cyclohexane (⑀ϭ 2.1, (28)). As the alkyl linker bridging the galactoside and the dansyl reporter of Dns 2 -S-Gal is short (2 carbons), the microenvironment probed should be in the immediate vicinity of the galactosyl binding site. On using analogs with increasing alkyl bridge lengths (Dns 3 -S-Gal or Dns 6 -S-Gal), the signal em [max] was progressively shifted toward higher wavelengths, suggesting that the dansyl is progressively displaced toward a less hydrophobic environment. In this context, Zani et al. (14) proposed a structural model of MelB consisting of a well-like structure open toward the periplasmic space and closed at the bottom by the cosubstrate binding and/or translocation domain in close contact with the cytoplasm. The data reported here might indicate that the pathway becomes less polar near the bottom of the well. Such a polarity gradient could vanish over a restricted distance and could partially shield the sugar ligands in the site from the solvent. Interestingly, a similar situation seems to prevail at the metal binding site of metaloprotroteins (29) or at the H ϩ binding site in the membrane sector of H ϩ -transporting ATPases (30). Yamashita et al. (29) pointed out that the metal ion binding sites of most metaloproteins are surrounded by an hydrophobic environment and introduced the concept of hydrophobic contrast. They derived an hydrophobic contrast function that can be used to predict the location of the metal binding site in metaloproteins structures. Importantly, they considered that the function describing the hydrophobic contrast in metal ion sites "may indirectly represent electrostatic and hydration components of the free energy of metal binding" (29). Extrapolating this notion to the binding of any hydrophilic substrate, we speculate that the hydrophobic contrast between the MelB sugar-binding site and its surroundings that is illustrated in this study has an analogous functional implication.
The Dns 2 -S-Gal signal amplitude is small and has a em [max] of about 490 nm in a nominally sodium-free medium, whereas the signal is much larger and has a em [max] of 465 nm in NaCl-containing media. The shift of em [max] toward a lower wavelength observed on adding NaCl suggests that the activating monovalent cation enhances the hydrophobicity of the microenvironment of Dns 2 -S-Gal bound to MelB. The resulting increase of the probe quantum yield accounts for at least part of the signal amplitude enhancement. The NaCl-induced change in Dns 2 -S-Gal microenvironment polarity most likely reflects a variation of MelB structure close to or at the sugar-binding site. This suggestion is consistent with previous evidence indicating that the structure of MelB is modified in the presence of Na ϩ ions (8), (16,17). Another interesting finding is that the ratio of the (Trp 3 Dns)-FRET and direct fluorescence signal is twice as large in the presence as in the absence of Na ϩ ion, suggesting that more light is absorbed by the acceptor when Na ϩ ions rather than H ϩ are bound to the transporter. This effect may be due to a change in the orientation of donor Trp(s) and/or of the acceptor Dns 2 -S-Gal caused by modification of their respective local environment and/or structure. Ioninduced modification of MelB structure could also change the donor(s)-acceptor distance. Better understanding of these spectroscopic events and possible structural implications requires the prior identification of the tryptophanyl residues acting as energy donors in the (Trp 3 Dns)-FRET phenomenon. In the accompanying paper (32), we used a site-directed mutagenesis approach to assess the participation of each of the eight Trps of MelB to the (Trp 3 Dns)-FRET signal.