In vivo and in vitro characterizations of melibiose permease (MelB) conformation-dependent nanobodies reveal sugar-binding mechanisms.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the Na+-coupled major facilitator superfamily transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well-studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelBSt by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelBSt under 4 ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelBSt and melibiose transport assays to determine the effects on MelBSt functions. We found that all selected Nbs showed partial to complete inhibitions of MelBSt transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelBSt/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelBSt complex retained binding to the coupling cation Na+ and also to the regulatory enzyme EIIAGlc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIAGlc/MelBSt complex also retained binding to Nb733 and formed a stable supercomplex. All data indicated that MelBSt trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIAGlc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology.

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane-based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine KM, Vmax, KD, and kobs values for different sugar substrates. KD and kobs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H+ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H+ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s-1) and for sugar translocation (2 s-1 - 30 s-1 for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.

The proton electrochemical gradient induces a kinetic asymmetry in the symport cycle of LacY.

LacY catalyzes accumulation of galactosides against a concentration gradient by coupling galactoside and H+ transport (i.e., symport). While alternating access of sugar- and H+-binding sites to either side of the membrane is driven by binding and dissociation of sugar, the electrochemical H+ gradient ([Formula: see text]) functions kinetically by decreasing the Km for influx 50- to 100-fold with no change in Kd The affinity of protonated LacY for sugar has an apparent pK (pKapp) of ∼10.5, due specifically to the pKa of Glu325, a residue that plays an irreplaceable role in coupling. In this study, rates of lactose/H+ efflux were measured from pH 5.0 to 9.0 in the absence or presence of a membrane potential (ΔΨ, interior positive), and the effect of the imposed ΔΨ on the kinetics of efflux was also studied in right-side-out membrane vesicles. The findings reveal that [Formula: see text] induces an asymmetry in the transport cycle based on the following observations: 1) the efflux rate of WT LacY exhibits a pKapp of ∼7.2 that is unaffected by the imposed ΔΨ; 2) ΔΨ increases the rate of efflux at all tested pH values, but enhancement is almost 2 orders of magnitude less than observed for influx; 3) mutant Glu325 - Ala does little or no efflux in the absence or presence of ΔΨ, and ambient pH has no effect; and 4) the effect of ΔΨ (interior positive) on the Km for efflux is almost insignificant relative to the 50- to 100-fold decrease in the Km for influx driven by ΔΨ (interior negative).

Arg302 governs the pKa of Glu325 in LacY.

Lactose permease is a paradigm for the major facilitator superfamily, the largest family of ion-coupled membrane transport proteins known at present. LacY carries out the coupled stoichiometric symport of a galactoside with an H+, using the free energy released from downhill translocation of H+ to drive accumulation of galactosides against a concentration gradient. In neutrophilic Escherichia coli, internal pH is kept at ∼7.6 over the physiological range, but the apparent pK (pKapp) for galactoside binding is 10.5. Surface-enhanced infrared absorption spectroscopy (SEIRAS) demonstrates that the high pKa is due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively. Deprotonation is also obligatory for turnover, however. Here, we utilize SEIRAS to study the effect of mutating residues in the immediate vicinity of Glu325 on its pKa The results are consistent with the idea that Arg302 (helix IX) is important for deprotonation of Glu325.

Engineered occluded apo-intermediate of LacY.

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

Oversized galactosides as a probe for conformational dynamics in LacY.

Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4-20 µM-1·s-1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or ß-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.

Crystal Structure of a ligand-bound LacY-Nanobody Complex.

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb-LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.

pKa of Glu325 in LacY.

Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and a H+ across the cytoplasmic membrane of Escherichia coli (galactoside/H+ symport). One of the most important aspects of the mechanism is the relationship between protonation and binding of the cargo galactopyranoside. In this regard, it has been shown that protonation is required for binding. Furthermore when galactoside affinity is measured as a function of pH, an apparent pK (pKapp) of ∼10.5 is obtained. Strikingly, when Glu325, a residue long known to be involved in coupling between H+ and sugar translocation, is replaced with a neutral side chain, the pH effect is abolished, and high-affinity binding is observed until LacY is destabilized at alkaline pH. In this paper, infrared spectroscopy is used to identify Glu325 in situ. Moreover, it is demonstrated that this residue exhibits a pKa of 10.5 ± 0.1 that is insensitive to the presence of galactopyranoside. Thus, it is apparent that protonation of Glu325 specifically is required for effective sugar binding to LacY.

Crystal structure of a LacY-nanobody complex in a periplasmic-open conformation.

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside-H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed. The structure confirms biochemical data indicating that the Nb binds stoichiometrically with nanomolar affinity to the periplasmic face of LacY primarily to the C-terminal six-helix bundle. The structure is novel because the pathway to the sugar-binding site is constricted and the central cavity containing the galactoside-binding site is empty. Although Phe27 narrows the periplasmic cavity, sugar is freely accessible to the binding site. Remarkably, the side chains directly involved in binding galactosides remain in the same position in the absence or presence of bound sugar.

YidC assists the stepwise and stochastic folding of membrane proteins.

How chaperones, insertases and translocases facilitate insertion and folding of complex cytoplasmic proteins into cellular membranes is not fully understood. Here we utilize single-molecule force spectroscopy to observe YidC, a transmembrane chaperone and insertase, sculpting the folding trajectory of the polytopic α-helical membrane protein lactose permease (LacY). In the absence of YidC, unfolded LacY inserts individual structural segments into the membrane; however, misfolding dominates the process so that folding cannot be completed. YidC prevents LacY from misfolding by stabilizing the unfolded state from which LacY inserts structural segments stepwise into the membrane until folding is completed. During stepwise insertion, YidC and the membrane together stabilize the transient folds. Remarkably, the order of insertion of structural segments is stochastic, indicating that LacY can fold along variable pathways toward the native structure. Since YidC is essential in membrane protein biogenesis and LacY is a model for the major facilitator superfamily, our observations have general relevance.

A chemiosmotic mechanism of symport.

Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and an H(+) across the Escherichia coli membrane (galactoside/H(+) symport). Initial X-ray structures reveal N- and C-terminal domains, each with six largely irregular transmembrane helices surrounding an aqueous cavity open to the cytoplasm. Recently, a structure with a narrow periplasmic opening and an occluded galactoside was obtained, confirming many observations and indicating that sugar binding involves induced fit. LacY catalyzes symport by an alternating access mechanism. Experimental findings garnered over 45 y indicate the following: (i) The limiting step for lactose/H(+) symport in the absence of the H(+) electrochemical gradient (∆µÌƒH+) is deprotonation, whereas in the presence of ∆µÌƒH+, the limiting step is opening of apo LacY on the other side of the membrane; (ii) LacY must be protonated to bind galactoside (the pK for binding is ∼10.5); (iii) galactoside binding and dissociation, not ∆µÌƒH+, are the driving forces for alternating access; (iv) galactoside binding involves induced fit, causing transition to an occluded intermediate that undergoes alternating access; (v) galactoside dissociates, releasing the energy of binding; and (vi) Arg302 comes into proximity with protonated Glu325, causing deprotonation. Accumulation of galactoside against a concentration gradient does not involve a change in Kd for sugar on either side of the membrane, but the pKa (the affinity for H(+)) decreases markedly. Thus, transport is driven chemiosmotically but, contrary to expectation, ∆µÌƒH+ acts kinetically to control the rate of the process.

Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIAGlc.

In a variety of bacteria, the phosphotransferase protein IIA(Glc) plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H(+), using a mechanism in which sugar- and H(+)-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIA(Glc) (inducer exclusion). Here we report the thermodynamic features of the IIA(Glc)-LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIA(Glc) binds to LacY with a Kd of about 5 µM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIA(Glc) binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIA(Glc) blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIA(Glc) binding. The precise mechanism of the inhibition of LacY by IIA(Glc) elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIA(Glc).

Transient conformers of LacY are trapped by nanobodies.

The lactose permease of Escherichia coli (LacY), a highly dynamic membrane protein, catalyzes symport of a galactopyranoside and an H(+) by using an alternating access mechanism, and the transport cycle involves multiple conformational states. Single-domain camelid nanobodies (Nbs) developed against a LacY mutant immobilized in an outward (periplasmic)-open conformation bind to the flexible WT protein and stabilize the open-outward conformation(s). Here, we use site-directed, distance-dependent Trp quenching/unquenching of fluorescent probes inserted on opposite surfaces of LacY to assess the conformational states of the protein complexed with each of eight unique Nbs that bind exclusively to the periplasmic side and block transport, but increase the accessibility of the sugar-binding site. Nb binding involves conformational selection of LacY molecules with exposed binding epitopes. Each of eight Nbs induces quenching with three pairs of cytoplasmic Trp/fluorophore probes, indicating closing of cytoplasmic cavity. In reciprocal fashion, the same Nbs induce unquenching of fluorescence in three pairs of periplasmic probes due to opening of the periplasmic cavity. Because the extent of fluorescence change with various Nbs differs and the differences correlate with changes in the rate of sugar binding, it is also concluded that the Nbs stabilize several different outward-open conformations of LacY.

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site.

The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.

An Asymmetric Conformational Change in LacY.

Galactoside/H+ symport by the lactose permease of Escherichia coli (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H+-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S-S bond restores opening of the periplasmic cavity and galactoside binding. Furthermore, nanobodies that stabilize the double-Cys mutant in a periplasmic-open conformation and allow free access of galactoside to the binding site do so only after reduction of the S-S bond. In contrast, when cross-linked LacY is solubilized in detergent, galactoside binding is observed, indicating that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site.

Real-time conformational changes in LacY.

Galactoside/H(+) symport across the cytoplasmic membrane of Escherichia coli is catalyzed by lactose permease (LacY), which uses an alternating access mechanism with opening and closing of deep cavities on the periplasmic and cytoplasmic sides. In this study, conformational changes in LacY initiated by galactoside binding were monitored in real time by Trp quenching/unquenching of bimane, a small fluorophore covalently attached to the protein. Rates of change in bimane fluorescence on either side of LacY were measured by stopped flow with LacY in detergent or in proteoliposomes and were compared with rates of galactoside binding. With LacY in proteoliposomes, the periplasmic cavity is tightly sealed and the substrate-binding rate is limited by the rate of opening of this cavity. Rates of opening, measured as unquenching of bimane fluorescence, are 20-30 s(-1), independent of sugar concentration and essentially the same in detergent or in proteoliposomes. On the cytoplasmic side of LacY in proteoliposomes, slow bimane quenching (i.e., closing of the cavity) is observed at a rate that is also independent of sugar concentration and similar to the rate of sugar binding from the periplasmic side. Therefore, opening of the periplasmic cavity not only limits access of sugar to the binding site of LacY but also controls the rate of closing of the cytoplasmic cavity.

Functional architecture of MFS D-glucose transporters.

The Major Facilitator Superfamily (MFS) is a diverse group of secondary transporters with over 10,000 members, found in all kingdoms of life, including Homo sapiens. One objective of determining crystallographic models of the bacterial representatives is identification and physical localization of residues important for catalysis in transporters with medical relevance. The recently solved crystallographic models of the D-xylose permease XylE from Escherichia coli and GlcP from Staphylococcus epidermidus, homologs of the human D-glucose transporters, the GLUTs (SLC2), provide information about the structure of these transporters. The goal of this work is to examine general concepts derived from the bacterial XylE, GlcP, and other MFS transporters for their relevance to the GLUTs by comparing conservation of functionally critical residues. An energy landscape for symport and uniport is presented. Furthermore, the substrate selectivity of XylE is compared with GLUT1 and GLUT5, as well as a XylE mutant that transports D-glucose.

Outward-facing conformers of LacY stabilized by nanobodies.

The lactose permease of Escherichia coli (LacY), a highly dynamic polytopic membrane protein, catalyzes stoichiometric galactoside/H(+) symport by an alternating access mechanism and exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a LacY mutant in an outward (periplasmic)-open conformation to stabilize this state of the WT protein. Twelve purified Nbs inhibit lactose transport in right-side-out membrane vesicles, indicating that the Nbs recognize epitopes on the periplasmic side of LacY. Stopped-flow kinetics of sugar binding by WT LacY in detergent micelles or reconstituted into proteoliposomes reveals dramatic increases in galactoside-binding rates induced by interaction with the Nbs. Thus, WT LacY in complex with the great majority of the Nbs exhibits varied increases in access of sugar to the binding site with an increase in association rate constants (kon) of up to ∼ 50-fold (reaching 10(7) M(-1) ⋅ s(-1)). In contrast, with the double-Trp mutant, which is already open on the periplasmic side, the Nbs have little effect. The findings are clearly consistent with stabilization of WT conformers with an open periplasmic cavity. Remarkably, some Nbs drastically decrease the rate of dissociation of bound sugar leading to increased affinity (greater than 200-fold for lactose).

Structure of sugar-bound LacY.

Here we describe the X-ray crystal structure of a double-Trp mutant (Gly46→Trp/Gly262→Trp) of the lactose permease of Escherichia coli (LacY) with a bound, high-affinity lactose analog. Although thought to be arrested in an open-outward conformation, the structure is almost occluded and is partially open to the periplasmic side; the cytoplasmic side is tightly sealed. Surprisingly, the opening on the periplasmic side is sufficiently narrow that sugar cannot get in or out of the binding site. Clearly defined density for a bound sugar is observed at the apex of the almost occluded cavity in the middle of the protein, and the side chains shown to ligate the galactopyranoside strongly confirm more than two decades of biochemical and spectroscopic findings. Comparison of the current structure with a previous structure of LacY with a covalently bound inactivator suggests that the galactopyranoside must be fully ligated to induce an occluded conformation. We conclude that protonated LacY binds D-galactopyranosides specifically, inducing an occluded state that can open to either side of the membrane.

Substrate-induced changes in the structural properties of LacY.

The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H(+) across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H(+)-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H(+)- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H(+) symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154→Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154→Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.