The lipid bilayer determines helical tilt angle and function in lactose permease of
            <i>Escherichia coli</i>

Coutre, Johannes le; Narasimhan, L. R.; Patel, C. K. N.; Kaback, H. Ronald

doi:10.1073/pnas.94.19.10167

Cited by 94 publications

(78 citation statements)

References 27 publications

(16 reference statements)

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“…1. The values were in the range from 0.01 to 0.02 lactose (min⅐permease) Ϫ1 and are in reasonable agreement with the rate of efflux previously observed at pH 6.0 in the presence of uncoupler (0.02 lactose (min⅐permease) Ϫ1 ) (12) and observed with a different permease preparation at pH 7.5 (22). These values are much slower than reported rates of proton pumping by bacteriorhodopsin (for example see Ref.…”

Section: Resultssupporting

confidence: 72%

A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease

Patzlaff¹,

Brooker²,

Barry³

2000

Journal of Biological Chemistry

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In chemiosmotic coupling, a transmembrane ion gradient is used as the source of energy to drive reactions. This process occurs in all cells, but the microscopic mechanism is not understood. Here, Escherichia coli lactose permease was used in a novel spectroscopic method to investigate the mechanism of chemiosmotic coupling in secondary active transporters. To provide a light-triggered electrochemical gradient, bacteriorhodopsin was co-reconstituted with the permease, and reaction-induced Fourier transform-infrared spectra were obtained from the co-reconstituted samples. The bacteriorhodopsin contributions were subtracted from these data to give spectra reflecting permease conformational changes that are induced by an electrochemical gradient. Positive bands in the 1765-1730 cm ؊1 region are attributable to carboxylic acid residues in the permease and are consistent with changes of pK a , protonation state, or environment. This is the first direct information concerning gradient-induced structural changes in the permease at the single amino acid level. Ultimately, these structural changes facilitate galactoside binding and may be involved in the storage of free energy.In secondary active transport, proteins employ a transmembrane electrochemical potential to translocate solutes across the membrane. This process plays a central metabolic role in all living cells. The molecular mechanism by which a transporter transduces a protonmotive force into a solute gradient is the focus of our study. The lactose permease is a transmembrane protein that is responsible for the uptake of lactose into the bacterial cytoplasm (1). The permease is a member of the major facilitator superfamily; members of the major facilitator superfamily catalyze uniport, symport, or antiport (reviewed in Ref.2). The lactose permease is a symporter that can harness the free energy stored in a proton electrochemical gradient to drive the accumulation of lactose against a concentration gradient (1). The permease transports protons and lactose with a one-to-one stoichiometry. A number of mutagenesis studies have led to models suggesting that a few ionizable residues along transmembrane segments play key roles in the mechanism of H ϩ /lactose symport (3, 4). However, direct evidence regarding the mechanism of chemiosmotic coupling in the lactose permease and related transporters has not been available. The goal of this study was to obtain dynamic information about structural changes in the permease associated with the imposition of an electrochemical gradient. To carry out this experiment, we have employed co-reconstitution (5-7) of the permease with a light-driven proton pump, bacteriorhodopsin, and reaction-induced FT-IR 1 spectroscopy (8). Reaction-induced FT-IR spectroscopy requires that a single sample be modulated between two structural states of interest. Co-reconstituted samples provide such a light-activatable and reversible experimental system. When a laser flash is given, bacteriorhodopsin pumps protons across the membrane and creates a H ϩ...

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Section: Resultssupporting

confidence: 72%

A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease

Patzlaff¹,

Brooker²,

Barry³

2000

Journal of Biological Chemistry

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“…[20][21][22][23][24]. It is also likely that the alternating access model for LacY involves formation of an occluded intermediate (25,26), which is consistent with the highly dynamic nature of the protein (15,(27)(28)(29)(30)(31).…”

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confidence: 50%

Role of the irreplaceable residues in the LacY alternating access mechanism

Zhou¹,

Jiang²,

Kaback

2012

Proc. Natl. Acad. Sci. U.S.A.

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Few side chains in the galactoside/H + symporter LacY (lactose permease of Escherichia coli) are irreplaceable for an alternating access mechanism in which sugar binding induces closing of the cytoplasmic cavity and reciprocal opening of a periplasmic cavity. In this study, each irreplaceable residue was mutated individually, and galactoside-induced opening or closing of periplasmic or cytoplasmic cavities was probed by site-directed alkylation. Mutation of Glu126 (helix IV) or Arg144 (helix V), which are essential for sugar binding, completely blocks sugar-induced periplasmic opening as expected. Remarkably, however, replacement of Glu269 (helix VIII), His322 (helix X), or Tyr236 (helix VII) causes spontaneous opening of the periplasmic cavity in the absence of sugar and decreased closing of the cytoplasmic cavity in the presence of galactoside. In contrast, mutation of Arg302 (helix IX) or Glu325 (helix X) has no such effect, and sugar binding induces normal opening and closing of periplasmic and cytoplasmic cavities. Possibly, Glu269, His322, and Tyr236 act in concert to coordinate opening and closing of the cavities by binding water, which also acts as a cofactor in H + translocation. Mutation of the triad results in loss of the bound water, which destabilizes LacY, and the cavities open and close in an uncoordinated manner. Thus, the triad mutants exhibit poor affinity for sugar, and galactoside/H + symport is abolished as well.membrane protein | membranes | protein dynamics | transport T he lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily of membrane transport proteins, catalyzes the coupled translocation of a galactoside and an H + (i.e., galactoside/H + symport). Thus, LacY transduces free energy stored in an H + electrochemical gradient (Δμ H +) into a galactoside concentration gradient. As transport is obligatorily coupled, LacY also transduces free energy stored in a sugar concentration gradient into Δμ H +, the polarity of which depends on the direction of the sugar gradient (reviewed in ref. 1). LacY has been solubilized from the membrane, purified to homogeneity in a highly functional state (2), and is structurally (3, 4), as well as functionally (5), a monomer.X-ray crystal structures of LacY (6-9) and various independent biochemical and spectroscopic findings (10-17) provide converging evidence for an alternating access mechanism. By this means, sugar binding induces coordinated opening and closing of periplasmic and cytoplasmic cavities, respectively, thereby allowing accessibility of sugar-and H + -binding sites to either side of the membrane (i.e., the alternating access model; reviewed in refs. 18, 19). A similar model has been proposed for a number of other transporters (e.g., refs. 20-24). It is also likely that the alternating access model for LacY involves formation of an occluded intermediate (25,26), which is consistent with the highly dynamic nature of the protein (15, 27-31).Cys-scanning and site-directed mutagenesis of each residue in LacY de...

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“…Some conformations may be more susceptible to inactivation than others. It has been suggested that membrane protein structure is significantly influenced by ordering imposed by the bilayer (17)(18)(19)(20). Thus, diminishing conformational restraints by extraction into a less ordered detergent micelle could increase conformational flexibility.…”

Section: Discussionmentioning

confidence: 99%

Building a Thermostable Membrane Protein

Zhou¹,

Bowie²

2000

Journal of Biological Chemistry

141

142

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The poor stability of membrane proteins in detergent solution is one of the main technical barriers to their structural and functional characterization. Here we describe a solution to this problem for diacylglycerol kinase (DGK), an integral membrane protein from Escherichia coli. Twelve enhanced stability mutants of DGK were obtained using a simple screen. Four of the mutations were combined to create a quadruple mutant that had improved stability in a wide range of detergents. In n-octylglucoside, the wild-type DGK had a thermal inactivation half-life of 6 min at 55°C, while the quadruple mutant displayed a half-life of 35 min at 80°C. In addition, the quadruple mutant had improved thermodynamic stability. Our approach should be applicable to other membrane proteins that can be conveniently assayed.Biochemical and structural studies of membrane proteins often require the proteins to be extracted from the lipid bilayer into a detergent micelle. Membrane proteins often exhibit unfavorable properties in detergent solution, however, such as poor solubility or stability (1, 2). Since the interactions between membrane proteins and detergents are very complex, the same protein can display different properties in different detergents. Thus, it is usually fruitful to screen for a detergent in which the protein of interest is active and stable. Unfortunately, many membrane proteins are only marginally stable and short-lived in even the best available detergent. In such cases, there are few alternatives for improving the experimental properties of the protein and the system can remain largely intractable.Recently, we discovered that single side-chain alterations can significantly improve the properties of the membrane protein diacylglycerol kinase (DGK) 1 from Escherichia coli, in detergent solution and that these stabilizing mutations can occur with high frequency. DGK is a 121-residue, trimeric enzyme, containing three transmembrane helices, that catalyzes the conversion of diacylglycerol and ATP to phosphatidic acid (3-7). We examined the stability of 20 different cysteine substitution mutants in DGK and found two mutations (I53C and I70C) that significantly enhanced the resistance of DGK to thermal inactivation. These results suggested that it might not be difficult to identify stabilizing mutations in a pool of random mutants (11). Here we show that enhanced stability mutants of DGK can be readily identified by screening a mutant library and that a robust membrane protein can be constructed by combining individual mutations. EXPERIMENTAL PROCEDURES Materials-The detergents n-octyl-␤-D-glucoside (OG), n-decyl-␤-Dmaltoside (DM), Cymal-5, and n-dodecyl-␤-D-maltoside(DDM)were obtained from Anatrace. LDAO and Empigen were obtained from Calbiochem. Cardiolipin and 1,2-sn-dioleoylglycerol (DAG) were obtained from Avanti Polar Lipids. All other chemicals were from Fisher or Sigma.Mutagenesis-We constructed a plasmid pSD005, containing a synthetic DGK gene. Plasmid pSD005 is identical to pSD004 (8), except that two mutatio...

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The lipid bilayer determines helical tilt angle and function in lactose permease of Escherichia coli

Cited by 94 publications

References 27 publications

A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease

A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease

Role of the irreplaceable residues in the LacY alternating access mechanism

Building a Thermostable Membrane Protein

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