Single-molecule FRET reveals sugar-induced conformational dynamics in LacY

Self Cite

The effect of bulk-phase pH on the apparent affinity (K d app ) of purified wild-type lactose permease (LacY) for sugars was studied. K d app values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. K d app for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high pK a of Ϸ10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/H ؉ symport in either direction across the membrane.lactose permease ͉ membrane transporters ͉ pH titrations ͉ proton translocation ͉ substrate affinity T he lactose permease of Escherichia coli (LacY), a paradigm for the major facilitator superfamily (MFS) of membrane transport proteins (1, 2), translocates a galactosidic sugar and an H ϩ across the cytoplasmic membrane (3, 4). This coupled translocation mechanism enables LacY to use free energy stored in an electrochemical H ϩ gradient (⌬ Hϩ ) to drive sugar accumulation against a concentration gradient (5-7).X-ray crystal structures of LacY (8-10), all of which are in an inward-facing conformation, and a wealth of biochemical/ biophysical data (7, 11-16) provide evidence for an alternatingaccess mechanism of action. By this means, upon sugar binding or imposition of a ⌬ Hϩ ) (interior negative and/or alkaline), the inward-facing cavity closes with opening of an outward-facing cavity, thereby allowing alternative accessibility of the sugarbinding site, which is at the apex of the cavity in the middle of the molecule, to either side of the membrane. A similar model has been proposed for the glycerol phosphate/phosphate antiporter GlpT, a related MFS protein (17) and the ABC transporter Sav 1866 (18). The alternating-access model involves a global conformational change, which is consistent with the highly dynamic nature of LacY (7,13,14,16,(19)(20)(21)(22)(23).A simple kinetic model for lactose/H ϩ symport (Fig. 1) has been proposed based on extensive studies of partial reactions (efflux, equilibrium exchange, and entrance counterflow) catalyzed by LacY and site-directed mutants defective in the symport mechanism (see ref. 5). Wild-type LacY is tightly coupled with respect to sugar and H ϩ translocation and does not translocate H ϩ without substrate or vice versa. The effect of ambient pH on ⌬ Hϩ -driven symport is bell-shaped with an optimum at approximately pH 7.5 (24, 25), whereas the partial reactions exhibit different dependences on pH. For example, rates of lac...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Protonation and sugar binding to LacY

Смирнова

Kasho²,

Kaback³

2008

Self Cite

“…Direct observation of distinct states of BBL in the transition region at equilibrium would provide compelling evidence for barrier limited folding (6,13). The current method of choice is single-molecule fluorescence resonance energy transfer (SM-FRET) experiments, which has been widely applied in the studies of protein folding, structure, and function, and is especially useful in detection of heterogeneity of populations (14)(15)(16)(17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

Direct observation of barrier-limited folding of BBL by single-molecule fluorescence resonance energy transfer

Huang

Ying

Fersht

2009

One controversial area in protein folding mechanisms is whether some small, ultra-fast-folding proteins exist in distinct native and denatured state ensembles, separated by an energy barrier, or if there is a continuum of states between native and denatured. In theory, the simplest way of distinguishing between single-state barrierless or ''downhill'' folding and conventional separate state folding is by single-molecule spectroscopy, which can detect either distinct populations of proteins or a continuum. But, the time resolution of approximately 1 ms of most confocal fluorescence microscopes for single-molecule fluorescence resonance energy transfer (SM-FRET) is longer than that for the structural relaxation of proteins such as BBL, whose mechanism of folding is controversial. We have constructed a highly sensitive confocal fluorescence microscope and measured the distribution of . The key protein in the unimodal hypothesis is BBL, a member of the peripheral subunit binding domain family, which is claimed to be a global downhill folder based especially on the dispersion of melting temperature for individual residues (2). It is extremely difficult to falsify unimodal folding because most equilibrium and kinetic data can be interpreted by numerous alternative mechanisms, especially when the rate of interconversion of D and N states is on a faster time scale than that of observation (6). The dependence of rates of folding and unfolding on denaturant concentration is good evidence for the nature of the mechanism-classical chevron plots with a steep limb are compatible with only a folding scenario with a free energy barrier and a transition state, so there is a significant change of solvent-accessible surface between the ground and transition states (6-8). That evidence combined with a rationalization of the anomalous behavior of BBL has been used as evidence that the folding of BBL can be accommodated by a barrier limited folding model, with some heterogeneity of the native state (9-12). Direct observation of distinct states of BBL in the transition region at equilibrium would provide compelling evidence for barrier limited folding (6, 13). The current method of choice is single-molecule fluorescence resonance energy transfer (SM-FRET) experiments, which has been widely applied in the studies of protein folding, structure, and function, and is especially useful in detection of heterogeneity of populations (14-21).We have applied SM-FRET to the B-domain of Protein A (BDPA) and directly observed the denatured and native states of this protein in its transition region (6), the signature of barrier-limited folding. We were not able to use SM-FRET to resolve unambiguously the folding mechanism of BBL because it folds in a fraction of a millisecond at room temperature, whereas the typical time resolution of SM-FRET was, until very recently, typically 1 ms (6,21,22), in which time BBL would rapidly equilibrate and appear as a time-averaged structure of the separate states if it folds by a barrier limited transition (23, 2...

“…Thus, measurement of interspin distances with nitroxide-labeled Cys pairs in LacY reveals that sugar binding induces a decrease in distances on the cytoplasmic side and a corresponding increase in distances on the periplasmic side (13,14). Site-directed alkylation of single Cys LacY mutants in either right-side-out membrane vesicles (15,16) or dodecyl-β-D-maltopyranoside (DDM) micelles (10), as well as single-molecule fluorescence (17) and thiol cross-linking (18), also indicates that sugar binding increases the probability of opening on the periplasmic side and closing on the cytoplasmic side.…”

mentioning

confidence: 99%

Real-time conformational changes in LacY

Смирнова¹,

Kasho²,

Kaback

2014

Self Cite

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. LacY is organized into two pseudosymmetrical bundles, each containing six transmembrane helices, most of which are irregular, with the N and C termini on the cytoplasmic side of the membrane. WT LacY and a conformationally restricted mutant exhibit an inward-facing conformation with a tightly sealed periplasmic side and a water-filled cavity open to the cytoplasm (6-9), which is the conformation present in the membrane in the absence of sugar (10). Another conformation has been observed recently with a double-Trp mutant (11) that exhibits an occluded galactoside molecule with a narrow opening on the periplasmic side and a tightly sealed cytoplasmic aspect (12) (Fig. 1A).LacY is highly dynamic and exhibits multiple conformations at any given time, and galactoside binding triggers a shift between conformers (1). Thus, measurement of interspin distances with nitroxide-labeled Cys pairs in LacY reveals that sugar binding induces a decrease in distances on the cytoplasmic side and a corresponding increase in distances on the periplasmic side (13,14). Site-directed alkylation of single Cys LacY mutants in either right-side-out membrane vesicles (15, 16) or dodecyl-β-D-maltopyranoside (DDM) micelles (10), as well as single-molecule fluorescence (17) and thiol cross-linking (18), also indicates that sugar binding increases the probability of opening on the periplasmic side and closing on the cytoplasmic side.Recently, conformational changes in LacY have been studied dynamically by site-directed Trp fluorescence quenching by a protonated His or Lys residue (19). Sugar binding leads to unquenching of Trp fluorescence in periplasmic LacY mutants N245W (helix VII) or F378...