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J. Biol. Chem., Vol. 279, Issue 47, 49214-49221, November 19, 2004

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Sugar Recognition by the Lactose Permease of Escherichia coli^*

José Luis Vázquez-Ibar, Lan Guan, Adam B. Weinglass, Gill Verner, Ruth Gordillo¶, and H. Ronald Kaback||

From the Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, Howard Hughes Medical Institute and the Chemistry and Biochemistry Department, UCLA, Los Angeles, California 90095-1662

Received for publication, July 2, 2004 , and in revised form, August 25, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity. The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site. We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269. Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates. Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N. It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The major facilitator superfamily (MFS)¹ is one of the largest families of membrane transport proteins found in archaeal, bacterial, and eukaryotic cell membranes (1) containing over 1000 members, some of which are clinically relevant (e.g. the glucose transporters). As revealed by phylogenetic analysis, most members of this family have a similar topology consisting of 12 transmembrane domains probably in -helical conformation, suggesting that they are related evolutionarily (1).

The lactose permease of Escherichia coli (LacY) is a paradigm for members of the MFS (2), which utilize free energy from an electrochemical proton gradient to catalyze the energetically uphill transport of D-galactopyranosides across the cell membrane or vice versa (3). Typical of the MFS, LacY contains 12 transmembrane helices connected by hydrophilic loops with both the N and C termini on the cytoplasmic face of the membrane (4–7), and it is functionally and structurally a monomer (7–9).

LacY is selective for disaccharides containing a D-galactopyranosyl ring, as well as D-galactose (10–12) but does not interact with D-glucopyranosides or D-glucose (12–14). Therefore, the substrate specificity of LacY is directed toward the galactopyranosyl ring of the substrate, and most importantly, toward the C-4 OH of the galactopyranosyl ring. Galactose is the most specific substrate of LacY but has a very low affinity (12), and substitutions at the anomeric position, particularly hydrophobic substitutions, markedly increase affinity by nonspecific interactions up to 3 orders of magnitude (15).

An important advance with regard to unraveling the mechanism of LacY has been the solution of the x-ray structure of the inward facing conformation of a LacY mutant (C154G) with a bound lactose homologue, -D-galactopyranosyl-1-thio--D-galactopyranoside (TDG) (7). The overall structure reveals a pseudo 2-fold symmetry between the N- and the C-terminal 6-helix domains, as proposed for the other members of the MFS (16). Remarkably, similar pseudo symmetry and helical packing was also found simultaneously in the x-ray structure of the P_i/glycerol-3-phosphate antiporter of E. coli (GlpT) (17), another MFS member. This structural similarity is even more interesting, because the two proteins have only 20% sequence identity and catalyze completely different reactions. Interestingly, LacY and GlpT were crystallized in the same inward facing conformation, although the GlpT structure was obtained in the absence of bound ligand.

The sugar binding site in LacY is located in a large water-filled cavity open only to the cytoplasm at the pseudo 2-fold axis of symmetry situated in the approximate middle of the membrane (see Fig. 1A). Arg-144 (helix V) and Glu-126 (helix IV) are the major determinants for sugar binding (see Fig. 1B). Arg-144 determines the stereoselectivity of LacY forming a bidentate H-bond with the O-3 and O-4 atoms of the galactopyranosyl ring, whereas Glu-126 probably interacts with O-4, O-5, or O-6 via water molecules. Interestingly, however, indirect biochemical approaches suggest the presence of a salt bridge between Glu-126 and Arg-144 when the substrate is not bound (18–22). Furthermore, as predicted (23), the structure exhibits a hydrophobic stacking between the galactopyranosyl ring of TDG and the indole side chain of Trp-151 (see Fig. 1B). The nature of this interaction has been extended recently (24) by luminescence spectroscopy.

View larger version (34K): [in this window] [in a new window]   FIG. 1. X-ray structure of LacY and binding site interactions of the galactopyranosyl ring. A, ribbon representation of the crystal structure of the C154G LacY mutant in an inward facing conformation with the bound lactose homologue, TDG (dark gray). The sugar binding site is present in a hydrophilic cavity at the pseudo 2-fold axis of symmetry at approximately the middle of the membrane. B, details of the important binding site interactions between TDG and LacY. The bottom of one galactopyranosyl ring (black) is within the van der Waal distance of Trp-151 (helix V), and Arg-144 (helix V) forms a bidentate H-bond with the O-3 and O-4 atoms of the galactopyranosyl ring. Also shown are Glu-269 (helix VIII), which appears to form a salt bridge with Arg-144 and is 3.4 Å from the indole N of Trp-151, and Glu-126 (helix IV), which may interact with the O-4, O-5, and/or O-6 atoms of the galactopyranosyl ring via water molecules. The data shown are derived from Ref. 7.

In this paper, we present experiments that describe the interplay between Trp-151 and Glu-269, which are involved in sugar recognition, and their implications with respect to coupling between substrate and H⁺ translocation. Examination of the luminescence properties of Trp-151 and fluorescence quenching experiments demonstrate that mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding properties of various sugars. Moreover, chemical modification of Trp-151 by N-bromosuccinimide (NBS) indicates that there is an H-bond between Glu-269 and the indole N of Trp-151 and that this interaction may orient both the sugar and the other essential side chains of the protein to form the binding site. Taken together, the findings support the contention that there is an H-bond between Glu-269 and Trp-151 that may rearrange during turnover.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Melibiose, TDG, potassium iodide, acrylamide, and N-bromosuccinimide, were purchased from Sigma. n-Dodecyl--D-maltopyranoside (DDM) was purchased from Calbiochem. Synthetic deoxyoligonucleotides were purchased from Sigma Genosys. Restriction endonucleases, T4 DNA ligase, and Vent DNA polymerase were purchased from New England Biolabs (Beverly, MA). All other materials were reagent grade and obtained from commercial sources.

Construction of LacY Mutants—Plasmid pCS19 (31) encoding single Trp-151 LacY and single Trp-151/C154G LacY containing a His₆ tag at the C terminus was constructed as described (24). Mutants at position 269 (E269A, E269D, E269Q, T266G/M267G/E269D) (25, 29) were subcloned into single Trp-151/C154G LacY as with KpnI/SpeI fragments. All constructs were confirmed by DNA sequencing.

Bacterial Strains, Growth, and LacY Purification—E. coli T184 (lacZ^-Y^-) transformed with pCS19 encoding each LacY mutant with a His₆ tag were grown in 6 liters of Luria-Bertani broth at 30 °C containing ampicillin (100 µg/ml) to an A₆₀₀ of 0.6 and induced with 0.5 mM isopropyl 1-thio--D-galactopyranoside. Cells were disrupted by passage through a French pressure cell, and the membrane fraction was harvested by ultracentrifugation. Membranes were solubilized by adding DDM to a final concentration of 2%, and LacY was purified by Co(II) affinity chromatography (Talon Superflow^TM, Palo Alto, CA) as described (7). Protein eluted with 200 mM imidazole was dialyzed against 20 mM Tris-HCl (pH 7.5), 0.008% DDM (for pCS19 mutants), concentrated by using a Vivaspin 20 concentrator (30-kDa cutoff; Vivascience, Germany), and stored on ice. As determined by sodium dodecylsulfate/12% polyacrylamide gel electrophoresis followed by silver nitrate staining, the preparations contained only a single band with a molecular mass of 33 kDa. Protein was assayed by using a micro-BCA kit (Pierce).

Transport Assays—E. coli T184 expressing given permease mutants were washed once with 100 mM potassium phosphate (potassium P_i,pH 7.5)/10 mM MgSO₄ and adjusted to an optical density of 10.0 at 600 nm (0.7 mg of protein/ml). Transport was initiated by the addition of [1-¹⁴C]lactose (5 mCi/mmol) to a final concentration of 0.4 mM. Samples were quenched at given times with 100 mM potassium P_i (pH 5.5)/100 mM LiCl and assayed by rapid filtration, as described (32).

Fluorescence Spectroscopy—Fluorescence was recorded in a SPEX Fluorolog 3 spectrofluorometer (Edison, NY) equipped with double-grating monochromators, Glan-Thompson polarizers, and a cuvette holder thermostated at 25 °C. Steady-state emission spectra and fluorescence quenching experiments were obtained in 20 mM Tris-HCl buffer (pH 7.5) containing 0.02% DDM using 5 µM final protein concentration with 0.3 x 1-cm quartz cuvettes. Trp was excited at 295 nm, and emission spectra were recorded at 1-nm intervals between 310 and 450 nm using 0.5-s integration times. In all cases, polarizers were used at the magic angle position (54.7°) to correct for polarization effects on intensity, reduce the direct contribution of light scattering, and eliminate polarization effects in monochromator transmittance (33, 34). Every spectrum is the average of at least two scans.

Fluorescence quenching experiments were performed by adding appropriate aliquots of stock solutions of 5 M potassium iodide or acrylamide to 200-µl samples containing 5 µM LacY. Concentrations of the quencher were varied from 0 to 300 mM. The data were analyzed by using Stern-Volmer plots for collisional quenching, F_o/F = 1 + K_SV[Q], where F_o and F correspond to the fluorescence emission of the protein in the absence and in the presence of a quencher at a given concentration [Q]. The slope of the plot F_o/F versus [Q] is the Stern-Volmer constant (K_SV), which is defined by the product of the bimolecular rate constant of the quenching process (k_q) and the fluorophore lifetime in the absence of quencher () (35). Because acrylamide absorbs weakly at 295 nm, fluorescence intensity was corrected as described (36).

Trp Oxidation by NBS—An aliquot (5 µl) from a freshly prepared stock solution of NBS in dimethyl sulfoxide (Me₂SO) was added to 1 ml of solution containing 3 µM DDM-solubilized LacY in 100 mM sodium acetate buffer (pH 5.0)/0.01% DDM. The final concentration of NBS in the sample was 200 µM. Because Trp oxidation leads to a complete loss of fluorescence, the extent of modification was measured by monitoring the decrease in fluorescence intensity with time until fluorescence reached a minimum. Experiments were performed in a 1-ml semimicro-quartz cuvette in a SPEX Fluorolog 3 spectrofluorometer, equipped with a cuvette holder thermostated at 25 °C. The excitation wavelength was set at 295 nm, and the emission wavelength was set between 335 and 345 nm, depending on the maximum emission wavelength of each mutant. It is not possible to obtain kinetics for the NBS-induced decrease in fluorescence by stop-flow, because NBS is highly labile in water, and LacY does not tolerate high concentrations of Me₂SO.

Phosphorescence Spectroscopy—Phosphorescence measurements were conducted at 77 K using a xenon-flash lamp as an excitation source. The excitation wavelength (280 nm) was selected with a monochromator (12-nm band pass), and the spectra were recorded between 390 and 490 nm at 1-nm intervals (5-nm band pass). Data were acquired with an initial delay of 0.1-ms, and every data point corresponds to the accumulation of 200 flashes. Measurements were made in a 5 mm x 5 mm quartz cuvette immersed in liquid N₂.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Single Trp-151 LacY Catalyzes Active Transport—To probe the environment of Trp-151 specifically, the other five Trp residues in LacY were replaced with Tyr (24). E. coli T184 (lacZ^-Y^-) expressing single Trp-151 LacY catalyzes lactose accumulation with a high rate to a steady state approaching that of wild-type LacY (Fig. 2).

View larger version (17K): [in this window] [in a new window]   FIG. 2. Transport properties of single Trp-151 LacY. Time courses of active transport by E. coli T184 (lacZ-Y-) expressing wild-type LacY (1), no LacY (2, pSC19 with no LacY insert), or single Trp-151 LacY (3). Aliquots (50 µl) of cell suspensions containing 35 µg of protein in 100 mM potassium Pi (pH 7.5)/10 mM MgSO4 were assayed at 0.4 mM final external concentration of lactose as described under "Experimental Procedures."

Trp luminescence has been used systematically as an internal reporter to study conformational rearrangements in proteins (34). It is well known that the emission properties of the indole ring are very sensitive to changes in the microenvironment (37). In addition, the nature and spatial disposition of the other amino acids around the aromatic ring can alter its emission properties (38). Therefore, the Trp-151 luminescence of LacY with single Trp at position 151 in the C154G background (24), with various replacements for Glu-269, was studied.

Single Trp-151/C154G demonstrates an emission maximum at 340 nm showing that the indole ring lies in a relatively aqueous environment, as indicated by the x-ray structure (Fig. 3) (24). Interestingly, all mutations of Glu-269 lead to alterations in the emission spectrum of Trp-151 (Fig. 3) and a loss of relative binding affinity (Fig. 3, inset). The conservative mutation E269D shifts the emission maximum to 345 nm and increases quantum yield, whereas affinity for ligand decreases 10-fold. Furthermore, consistent with previous work demonstrating that the introduction of a double Gly mutation (T266G/M267G) rescues both binding affinity and the ability of E269D to catalyze a transport (29), the same double Gly mutation in the single Trp-151/C154G/E269D background shifts the emission maximum and quantum yield back to that of single Trp-151/C154G. Two more mutations at position 269 (E269Q and E269A) lead to a complete loss of substrate binding, quenching of Trp-151 fluorescence, and a 4- or 6-nm shift in the emission spectrum to 336 and 334 nm, respectively.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Effect of Glu-269 substitutions on the emission spectrum of Trp-151 and substrate affinity. Emission spectra of purified, detergent-solubilized single Trp-151/C154G LacY (5 µM). Each spectrum was recorded using an excitation wavelength of 295 nm following the experimental conditions described under "Experimental Procedures." Substrate affinities of Glu-269 mutants are represented in the inset (25, 28, 29).

View larger version (21K): [in this window] [in a new window]   FIG. 4. Effect of Glu-269 mutations on Trp-151 oxidation by NBS. A, reaction mechanism of tryptophan oxidation by NBS. The 2 position of the indole ring is brominated (I–II). Subsequently, 2-haloindole is hydrolyzed to obtain the final oxindole (II–IV). B, NBS modification of Glu-269 mutants. The reaction is initiated by adding 200 µM NBS to each purified mutant. Excitation and emission wavelengths of 295 and 335–345 nm, respectively, were set depending on the maximum emission wavelength of each mutant.

Role of Trp-151 in Substrate Binding—Both x-ray (7) and luminescence (24) studies demonstrate that the galactopyranosyl moiety of TDG interacts hydrophobically with Trp-151. By monitoring the oxidation of Trp-151 by NBS, it is possible to examine how different substrates interact with Trp-151.

Consistent with the notion that the substrate reduces the reactivity of Trp-151 with NBS, the pre-incubation of single Trp-151/C154G LacY with TDG leads to a dramatic decrease in the reactivity of NBS with Trp-151 (Fig. 5A) compared with glucose, which does not bind to LacY. A saturating concentration of another LacY substrate (melibiose) is significantly less effective in blocking the reaction of Trp-151 with NBS. An alternative approach to directly examining hydrophobic stacking between Trp-151 and sugar is to follow spectral shifts in the 0,0 vibronic band of the Trp phosphorescence spectrum to longer wavelengths (24, 42, 43). Likewise, this approach illustrates that although TDG leads to a 4-nm red shift, melibiose leads to only a 2-nm red shift in the phosphorescence spectrum of Trp-151 (Fig. 5B).

View larger version (17K): [in this window] [in a new window]   FIG. 5. Interaction of substrate with Trp-151. A, time course of NBS modification of Trp-151. The oxidation of Trp-151 by NBS in the absence (no sugar) and presence of saturating concentrations of the indicated sugar was monitored in purified single Trp-151/C154G (3 µM) in DDM. Excitation and emission wavelengths were set to 295 and 340 nm, respectively. B, substrate effects on Trp-151 phosphorescence spectra. Phosphorescence spectra (at 77 K) of purified, DDM-solubilized single Trp-151/C154G LacY (10 µM) were recorded in the absence or presence of a given sugar at saturating concentration. The excitation wavelength was 280 nm, and the spectra were recorded as described under "Experimental Procedures."

View larger version (14K): [in this window] [in a new window]   FIG. 6. Comparing the environment of Trp-151 in single Trp-151/C154G LacY with single Trp-151 LacY. A, effect of the C154G mutation on the emission spectra of Trp-151. Emission spectrum of DDM-solubilized single Trp-151/C154G LacY (5 µM, C154G) and single Trp-151 LacY (5 µM, Cys-154 (C154)). Spectra were recorded using an excitation wavelength of 295 nm as described under "Experimental Procedures." B, effect of the C154G mutation on Trp-151 oxidation by NBS. Time course of Trp-151 oxidation in DDM-solubilized single Trp-151 LacY (3 µM) in the absence (Cys-154 (C154)) and presence of the C154G mutation. The excitation wavelength was 295 nm, and the emission wavelength was set at the maximum wavelength emission of each mutant.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The x-ray structure of an inward facing conformation of LacY with a bound substrate (7) provides a framework for understanding the molecular basis of substrate recognition and translocation by LacY, a paradigm for the MFS family of secondary membrane transport proteins. By integrating the structure with biochemical and biophysical analyses, a model for the mechanism for lactose/H⁺ symport has been proposed (7). Influx consists of six steps starting from the outward facing conformation (as shown in Fig. 7, state I), (i) protonation of LacY, (ii) binding of lactose, (iii) a conformational change that results in the inward facing conformation, (iv) release of substrate, (v) release of proton, and (vi) return to the outward facing conformation. Utilizing the x-ray structure of the inward facing conformation with substrate bound as a structural foundation, luminescent techniques have been exploited to gain detailed mechanistic insights into specific states and their transitions in the overall reaction.

View larger version (25K): [in this window] [in a new window]   FIG. 7. A working mechanism for lactose/H+ symport. N- and C-terminal domains are shown as ovals. Key residues are labeled; hydrogen bonds are shown as filled lines. The proton and substrate are shown as circles. There are six states in the translocation cycle (numbered I–VI). Modifications to the proposed mechanism (7) are the introduction of an H-bond between Trp-151 and Glu-269 in states III, IV, V, and VI (dashed black line).

Shortening (E269D), removing (E269A, E269Q), or manipulating (T266G/M267G/E269D) the carboxyl side chain at position 269 results in dramatic changes in the Trp-151 fluorescence emission spectra of Glu-269 mutants that correlate well with observed changes in the substrate binding affinity (25, 29). Therefore, it is strongly suggested that the H-bond observed between these two residues in the x-ray structure of the inward facing conformation with the ligand bound is also present in the absence of a ligand (Fig. 7, states V and/or VI). Moreover, most likely, subtle changes in the precise interaction of Trp-151 with Glu-269 translate into reorganization of essential residues in the substrate binding site, particularly Arg-144, leading to large changes in substrate affinity and specificity. Unfortunately, hydrophobic stacking of the substrate with the indole ring of Trp-151 precludes experiments aimed at determining how substrate binding influences H-bonding between Trp-151 and Glu-269.

Similarly, the observation that the reactivity of Trp-151 with NBS correlates with the ability of the side chain at position 269 to bind substrate (i.e. Glu-269 > Asp-269 > Gln-269) supports the notion that the modification of an indole group by NBS is increased when the NH group participates in an H-bond. Notably, the hydrophilic environment of Trp-151 and its reactivity with NBS appears to be dependent on the presence of a carboxyl group on the side chain of position 269. Structurally, an electrostatic interaction between the carboxyl group at position 269 and Arg-144 (Fig. 1B) may be critical in enabling Trp-151 to H-bond with Glu-269 and maintain the hydrophilic environment of Trp-151. However, a recent study suggesting that the environment of Arg-144 is influenced heavily by Glu-126 rather than Glu-269 (45) is consistent with other biochemical and biophysical approaches indicating that Arg-144 is charge-paired to Glu-126 in the absence of a substrate (19–22, 46). However, each of these interactions may be dynamic even in the C154G mutant, which catalyzes transport at a very low rate.

The double Gly mutant exemplifies the complexity of interactions in the substrate binding site. Although the double Gly mutation improves TDG binding affinity and lactose transport considerably with respect to E269D, the properties are by no means near wild type. First, wild type binds substrate with an apparent K_i of 48 µM, and the double Gly mutant exhibits an apparent K_i of 322 µM. Second, the apparent K_m of the double Gly mutant is increased 5-fold and the V_max is reduced 2-fold compared with wild type (29), and third, although influx is improved dramatically in the double Gly mutant, efflux is not. Most likely, the double Gly replacement partially rescues mutant E269D by altering the conformational dynamics of a localized region of helix VIII (29), enabling Asp-269 to contribute to the network of essential residues governing substrate affinity and specificity by influencing Trp-151 and Arg-144. However, subtle differences in the precise positioning of the carboxyl group at position 269 in the double Gly mutant compared with Glu-269 probably explain how the changes in fluorescence and quenching can be "like" wild type, whereas there is no improvement in H-bonding with Trp-151 as assessed by NBS modification.

The molecular mechanism of LacY involves a series of conformational changes coupling protonation to substrate binding and translocation (Fig. 7). However, the x-ray structure of the inward facing conformation of LacY and the Glu-269 mutants used in this study were obtained and characterized, respectively, on a C154G background that presumably represents state IV. Because C154G LacY exhibits a dramatic reduction in the rate of ligand-induced conformational changes relative to the wild-type LacY (44), although these studies indicate that Glu-269 is H-bonded with Trp-151 in the absence or presence of ligand, they do not provide insight into the environment and H-bonding state of Trp-151 and Glu-269 during other stages of the translocation cycle.

In particular, a mutational analysis of wild-type LacY demonstrates that Glu-269 and His-322 are both necessary for high affinity binding (28, 47), and studies of Mn(II) binding to mutant E269H/H322 (27) reveal that these two residues come into close proximity, perhaps allowing the imidazole group of His-322 to perturb the pK_a of Glu-269, thereby permitting protonation of the outward facing conformation at physiological pH (Fig. 7, states I and II) (47). However, in the x-ray structure of C154G, the substrate binding site of LacY is 5.8 Å away from His-322. These observations are consistent with the demonstration that one face of helix VIII rotates upon substrate binding (48) permitting Glu-269 to rearrange and potentially interact with both Trp-151 and His-322 at different stages in the translocation cycle. Critically, by comparing the environment of Trp-151 in the absence or presence of the C154G mutation, it is observed that Trp-151 becomes more accessible to an aqueous environment and interacts less efficiently with Glu-269 (Fig. 6). These findings are consistent with the interpretation that Glu-269 alternates between different conformations, interacting with either Trp-151 or His-322.

With respect to substrate binding to the outward facing conformation of LacY (Fig. 7, states II and III), it is likely that Glu-269 H-bonds to Arg-144 and Trp-151 for binding to occur. However, as shown in state III, Glu-269 is H-bonded to His-322. Therefore, during substrate binding, it is suggested that there is a coordinated rearrangement in the interactions between irreplaceable residues involved in sugar binding and proton translocation that may trigger the transition to the inward facing conformation. In other words, sugar binding to LacY may involve a dynamic process coupled with a conformational transition to the inward facing conformation.

	FOOTNOTES

 
^* This work was supported in part by Grant DK51131:09 from the National Institutes of Health (to H. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a Postdoctoral Fellowship from the Ministerio de Educacion Cultura y Deporte, Spain.

^|| To whom correspondence should be addressed: HHMI/UCLA, 5-748 MacDonald Research Laboratories, Box 951662, Los Angeles, CA 90095-1662. Tel.: 310-206-5053; Fax: 310-206-8623; E-mail: RonaldK{at}HHMI.UCLA.edu.

¹ The abbreviations used are: MFS, major facilitator superfamily; LacY, lactose permease; DDM, n-dodecyl--D-maltopyranoside; K_SV, Stern-Volmer quenching constant; NBS, N-bromosuccinimide; TDG, -D-galactopyranosyl-1-thio--D-galactopyranoside.

² N. Ermolova, I. Smirnova, V. Kasho, and H. R. Kaback, manuscript in preparation.

	ACKNOWLEDGMENTS

 
We thank Michael Ehrmann for generously providing plasmid pCS19 and we thank Alexandre Specht for insightful discussions.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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