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• W1968204553 abstract "In the preceding two papers (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem.272, 17605–17609; Hall, J. A., Thorgeirson, T. E., Liu, J., Shin, Y.-E., and Nikaido, H. (1997) J. Biol. Chem.272, 17610–17614), we showed that ligands that bind to theEscherichia coli maltose-binding protein (MBP) without producing the closure of its two lobes are not transported into the cytoplasm. Here, we examine various combinations of ligands, MBPs, and membrane-associated transporters, by utilizing reconstituted proteoliposomes, right side-out membrane vesicles, and intact cells. Closed forms of wild type MBP, complexed with maltose or maltodextrins, interacted with wild type transporter complex to stimulate the hydrolysis of ATP by MalK ATPase located on the other side of the membrane, as shown earlier for the maltose-MBP complex (Davidson, A. L., Shuman, H. A., and Nikaido, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2360–2364). In contrast, open forms of liganded MBPs, such as the complex containing wild type MBP and reduced, oxidized, or cyclic maltodextrins or the complex containing the mutant MBP MalE254 and unmodified maltodextrins, did not stimulate ATP hydrolysis, suggesting that the proper interaction between the ligand-MBP complex and the external surface of the transporter requires the former to be in the closed conformation. However, when a mutant transporter containing MalG511 was used, the already significant basal level of ATP hydrolysis was further stimulated not only by ligand MBPs in the closed form but also by those in the open form (except that containing β-cyclodextrin), data suggesting that the mutant transporter does not always require the closed MBP complex presumably because of its exceptionally strong affinity to MBP, described earlier (Dean, D. A., Hor, L.-I., Shuman, H. A., and Nikaido, H. (1992)Mol. Microbiol. 6, 2033–2040). Furthermore, this mutant transporter was able to transport reduced maltodextrin, and cells expressing the transporter were able to grow by using reduced maltodextrin, if the periplasmic concentrations of MBP were kept low so as not to inhibit the transport process. In the preceding two papers (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem.272, 17605–17609; Hall, J. A., Thorgeirson, T. E., Liu, J., Shin, Y.-E., and Nikaido, H. (1997) J. Biol. Chem.272, 17610–17614), we showed that ligands that bind to theEscherichia coli maltose-binding protein (MBP) without producing the closure of its two lobes are not transported into the cytoplasm. Here, we examine various combinations of ligands, MBPs, and membrane-associated transporters, by utilizing reconstituted proteoliposomes, right side-out membrane vesicles, and intact cells. Closed forms of wild type MBP, complexed with maltose or maltodextrins, interacted with wild type transporter complex to stimulate the hydrolysis of ATP by MalK ATPase located on the other side of the membrane, as shown earlier for the maltose-MBP complex (Davidson, A. L., Shuman, H. A., and Nikaido, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2360–2364). In contrast, open forms of liganded MBPs, such as the complex containing wild type MBP and reduced, oxidized, or cyclic maltodextrins or the complex containing the mutant MBP MalE254 and unmodified maltodextrins, did not stimulate ATP hydrolysis, suggesting that the proper interaction between the ligand-MBP complex and the external surface of the transporter requires the former to be in the closed conformation. However, when a mutant transporter containing MalG511 was used, the already significant basal level of ATP hydrolysis was further stimulated not only by ligand MBPs in the closed form but also by those in the open form (except that containing β-cyclodextrin), data suggesting that the mutant transporter does not always require the closed MBP complex presumably because of its exceptionally strong affinity to MBP, described earlier (Dean, D. A., Hor, L.-I., Shuman, H. A., and Nikaido, H. (1992)Mol. Microbiol. 6, 2033–2040). Furthermore, this mutant transporter was able to transport reduced maltodextrin, and cells expressing the transporter were able to grow by using reduced maltodextrin, if the periplasmic concentrations of MBP were kept low so as not to inhibit the transport process. Maltose and maltodextrins are transported in Escherichia coli by an ATP-dependent process, which requires the interaction between soluble, liganded maltose-binding protein (MBP) 1The abbreviations used are: MBP, maltose-binding protein; IPTG, isopropyl-β-d-galactoside. 1The abbreviations used are: MBP, maltose-binding protein; IPTG, isopropyl-β-d-galactoside. and a membrane-associated transporter (MalFGK2) composed of one copy each of MalF and MalG channel proteins and two copies of MalK ATPase (1Boos W. Lucht J.M. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 1175-1209Google Scholar, 2Davidson A.L Nikaido H. J. Biol. Chem. 1991; 266: 8946-8951Abstract Full Text PDF PubMed Google Scholar). The participation of MBP is absolutely needed for this transport process, and one of the reasons may be that the liganded MBP, by binding to the external surface of the transporter, sends a transmembrane signal so that the MalK ATPase can become activated on the opposite, internal surface of the membrane (3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google Scholar). The requirement for MBP, however, can be circumvented in “MBP-independent” transporter mutants, such as the one containing mutant MalG511 (MalFG511K2) (4Treptow N.A. Shuman H.A. J. Bacteriol. 1985; 163: 654-660Crossref PubMed Google Scholar), which constitutively hydrolyze ATP even in the absence of the liganded MBP (3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google Scholar). We have shown in the preceding papers (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 6Hall J.A. Thorgeirson T.E. Liu J. Shin Y.-E. Nikaido H. J. Biol. Chem. 1997; 272: 17610-17614Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) that MBP can bind its ligands in two different ways. Maltose and linear maltodextrins bind to MBP in a way that produces a slight red shift of the intrinsic fluorescence emission spectrum of the protein (called R mode (forred shift)) (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 7Szmelcman S. Schwartz M. Silhavy T.J. Boos W. Eur. J. Biochem. 1976; 65: 13-19Crossref PubMed Scopus (195) Google Scholar) and a characteristic hypochromatic trend in the <265-nm region of UV absorbance spectrum (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8Gehring K. Bao K. Nikaido H. FEBS Lett. 1992; 300: 33-38Crossref PubMed Scopus (11) Google Scholar). An earlier study showed that the binding of maltose and α-anomers of maltodextrins produced a large upfield shift of the NMR resonance of3H on the anomeric carbon of the reducing glucose residue (“end-on” mode) (9Gehring K. Williams P.G. Pelton J.G. Morimoto H. Wemmer D.E. Biochemistry. 1991; 30: 5524-5531Crossref PubMed Scopus (46) Google Scholar), and the R and end-on modes appear to refer to the same manner of ligand binding. In contrast, when the MBP binds β-cyclodextrin, or reduced or oxidized derivatives of maltodextrins, the fluorescence emission spectrum is blue-shifted (B mode (forblue shift)), and the UV absorbance differential spectra show no hypochromatic trend in the <265-nm region (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 8Gehring K. Bao K. Nikaido H. FEBS Lett. 1992; 300: 33-38Crossref PubMed Scopus (11) Google Scholar). Since β-cyclodextrin and the modified dextrins just mentioned do not contain a reducing sugar residue with its anomeric carbon, we hypothesize that this mode corresponds to the binding mode that does not involve the tight interaction of the hydrogen on the anomeric carbon with the binding site of MBP, i.e. the middle mode, earlier observed by 3H NMR for β-anomers of maltodextrins (9Gehring K. Williams P.G. Pelton J.G. Morimoto H. Wemmer D.E. Biochemistry. 1991; 30: 5524-5531Crossref PubMed Scopus (46) Google Scholar). Interestingly, a mutant MalE254 MBP, which allows the transport of maltose but not of maltodextrins (10Wandersman C. Schwartz M. Ferenci T. J. Bacteriol. 1979; 140: 1-13Crossref PubMed Google Scholar), binds unaltered maltodextrins exclusively via the B mode (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). By using representative ligands, we showed further that the two lobes of MBP become closed when the R mode binding occurs, whereas there is little or no closing of the lobes when the binding occurs through the B mode (6Hall J.A. Thorgeirson T.E. Liu J. Shin Y.-E. Nikaido H. J. Biol. Chem. 1997; 272: 17610-17614Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). There are some hints that the B mode binding may not lead to the successful transport of ligands through the MalFGK2complex. For example, maltodextrin derivatives that are reduced, oxidized, or substituted at their reducing glucose units bind to wild type MBP exclusively via the B mode as described above, and are not transported by the wild type E. coli (11Ferenci T. Muir M. Lee K.-S. Maris D. Biochim. Biophys. Acta. 1986; 860: 44-50Crossref PubMed Scopus (43) Google Scholar). The mutant MalE254 MBP, which binds unmodified maltodextrins exclusively via the B mode (see above), does not allow the transport of these ligands (10Wandersman C. Schwartz M. Ferenci T. J. Bacteriol. 1979; 140: 1-13Crossref PubMed Google Scholar). The failure to be transported can result either because the B mode complex fails to stimulate the ATPase activity of the transporter or because the B mode complex cannot deliver the ligand into the transport channel. The present paper examines this question and shows that the stimulation of ATP hydrolytic functions of the wild type MalFGK2 transporter is not induced by B mode ligand-MBP complex. We also show that ligands bound via the B mode to MBP are transported nevertheless through the mutant MalFG511K2complex and that these mutant cells are indeed able to grow by using reduced maltodextrins as their carbon and energy source. These are shown in TableI. Strains HN889, HN892, HN924, and HN930 were constructed by transferring the F′ factor carrying thelacI q mutation and Tn5 (kanamycinr) from HN596 to HN933, NT411 containing pLH16, NT229 containing pLH16, and HN931, respectively, using standard mating technique (17Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 82-85Google Scholar). High affinity lac repressor in these strains was necessary to decrease the level of transcription of malE alleles under control of the lacUV5 promoter (18Rasmussen B.A. MacGregor C.H. Ray P.H. Bassford P.J. J. Bacteriol. 1985; 164: 665-673Crossref PubMed Google Scholar) to a minimum when uninduced. Antibiotic concentrations added to media were as follows: streptomycin, 25 μg/ml; ampicillin, 100 μg/ml; and kanamycin, 50 μg/ml.Table IE. coli strains and plasmids used in this studyStrain or PlasmidGenotypeReference or SourceHS2019F− araΔ139 ΔlacU169 rpsL thi ΔmalE44412Shuman H.A. J. Biol. Chem. 1982; 257: 5455-5461Abstract Full Text PDF PubMed Google ScholarHN741argH his rpsL1 malTc ΔmalB13 ΔuncBC ilv::Tn10/F′ lacIqTn5 3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google ScholarHS3368F−Δ(argF-lac)U169 rpsL150 relAI rbsR flbB5301 ptsF25 thi-1 deoC1 malTc-1 Φ(malP-lacZ + )/lacY:Tn9H. A. ShumanNT229F− Δ(argF-lac)U169 araD139 rpsL150 thi-1 flbB5301 ptsF25 relA1 ΔmalE444 malG511 4Treptow N.A. Shuman H.A. J. Bacteriol. 1985; 163: 654-660Crossref PubMed Google ScholarNT411NT229 malTc-1 Φ(malP-lacZ + ) 4Treptow N.A. Shuman H.A. J. Bacteriol. 1985; 163: 654-660Crossref PubMed Google ScholarHN596malTcaraD lac rpsL1/F′(lacIq lacZ::Tn5proA + proB + )13Davidson A.L. Nikaido H. J. Biol. Chem. 1990; 265: 4254-4260Abstract Full Text PDF PubMed Google ScholarHN889NT411 (pJF2)/F′(lacIq lacZ::Tn5proAB + )This workHN892NT411 (pLH16)/F′(lacIq lacZ::Tn5proAB + )This workHN924NT229 (pLH16)/F′(lacIq lacZ::Tn5proAB + )This workHN930NT229 (pJF2)/F′(lacIq lacZ::Tn5proAB + )This workHN931NT229 (pJF2)This workHN932NT229 (pPD1)This workHN933NT411 (pJF2)This workHN934NT411 (pPD1)This workpEH1pBluescript S/K+ withmalE254 insertThis workpFG23malF malG bla13Davidson A.L. Nikaido H. J. Biol. Chem. 1990; 265: 4254-4260Abstract Full Text PDF PubMed Google ScholarpLH33malF malG511 bla 3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google ScholarpMR11malK cat14Reyes M. Shuman H.A. J. Bacteriol. 1988; 170: 4598-4602Crossref PubMed Google ScholarpPD1malE + (under pmalB control)bla15Duplay P. Bedouelle H. Fowler A. Zabin I. Saurin W. Hofnung M. J. Biol. Chem. 1984; 259: 10606-10613Abstract Full Text PDF PubMed Google ScholarpJF2malE + (under placUV5 control) bla16Fikes J.D. Bassford P.J. J. Bacteriol. 1987; 169: 2352-2359Crossref PubMed Google ScholarpLH16malE632 (under placUV5 control)blaH. A. Shuman Open table in a new tab Nonradioactive sugars have been described (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). [14C]Maltose was purchased from either Amersham Corp. or ICN Biomedicals, Inc. [3H]Maltotetraose was prepared at the National Tritium Labeling Facility from maltotetraose by exchange with tritium gas in the presence of palladium on BaSO4 (9Gehring K. Williams P.G. Pelton J.G. Morimoto H. Wemmer D.E. Biochemistry. 1991; 30: 5524-5531Crossref PubMed Scopus (46) Google Scholar). The specific activity of [3H]maltotetraose was estimated by assuming complete recovery of the sugar from the labeling reaction as well as by NMR. [3H]Maltotetraitol was made by reducing [3H]maltotetraose with sodium borohydride as described (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Wild type MBP and malE254 MBP were prepared as described (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), except that malE254 MBP was exclusively from strain HS2019 containing pEH1. Bound maltose was removed from malE254 MBP by extensive dialysis as described by Silhavy et al. (19Silhavy T.J. Szmelcman S. Boos W. Schwartz M. Proc. Nat. Acad. Sci. U. S. A. 1975; 72: 2120-2124Crossref PubMed Scopus (120) Google Scholar), and from wild type MBP by denaturation in 6 m guanidine HCl followed by renaturation through dialysis. Fluorescence emission spectroscopy was used to show that both the mutant and wild type MBPs were free of maltose and active (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The transporter complex (MalFGK2 or MalFG511K2) was prepared from strain HN741 containing pMR11 and either pFG23 or pLH33. Cells were grown, induced, and harvested as described (3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google Scholar). Total membrane fractions were prepared as described (2Davidson A.L Nikaido H. J. Biol. Chem. 1991; 266: 8946-8951Abstract Full Text PDF PubMed Google Scholar). Membrane proteins were solubilized by keeping vesicles on ice for 30 min in 20 mm KPO4, pH 6.2, 20% glycerol, 5 mm MgCl2, 1 mm dithiothreitol, 1.1% octyl-β-d-glucopyranoside (membrane protein:octyl-β-d-glucopyranoside ratio was about 3:1). This mixture was centrifuged at 160,000 × g for 1 h at 4 °C, and the supernatant was aliquoted and stored at −70 °C until use. Membrane vesicles containing the MalG511 transporter that were destined for use in the proteoliposome substrate uptake assay were solubilized as above except that sonicated E. coli acetone/ether-washed total lipids (Avanti Polar Lipids, Inc.) were added to a final concentration of 8–9 mg/ml. This mixture was then processed as above, and the supernatant fraction was used immediately. The affinities were determined from the concentration dependence of the quenching of the intrinsic protein fluorescence (7Szmelcman S. Schwartz M. Silhavy T.J. Boos W. Eur. J. Biochem. 1976; 65: 13-19Crossref PubMed Scopus (195) Google Scholar), measured on a Perkin-Elmer MPF-44B spectrofluorometer. The MBPs (0.2 μm) were dissolved in 10 mm KPO4, pH 7.0. Excitation was at 280 nm with a bandwidth of 7 nm, and emission was recorded at 348 and 346 nm for wild type and malE254 MBPs, respectively, with a bandwidth of 7 nm. Solubilized membrane proteins were reconstituted into liposomes via a detergent dilution method (13Davidson A.L. Nikaido H. J. Biol. Chem. 1990; 265: 4254-4260Abstract Full Text PDF PubMed Google Scholar). In all experiments 4.5 mg of sonicated E. coliacetone/ether-washed total lipids (Avanti Polar Lipids, Inc.) in 20 mm KPO4, pH 6.2, 2 mmβ-mercaptoethanol was mixed with 90 μg of solubilized proteins, and octyl-β-d-glucopyranoside was added to a final concentration of 1.1%. This mixture (0.54 ml) was then incubated on ice for 30 min, diluted into 14 ml of 20 mmKPO4, pH 6.2, 1 mm dithiothreitol, and centrifuged at 160,000 × g for 1 h at 4 °C to isolate the proteoliposomes. MBP was added prior to dilution to give a final concentration of 1.5 μm (for assays using wild type MBP) or 0.15 μm (for assays using malE254 MBP) after dilution. In these experiments, the weight ratio of E. coli phospholipids, MBP, and solubilized protein was 50:8:1 when wild type MBP was used and 50:1:1 when MalE254 MBP was used. Also, transport substrate was added prior to dilution to give a final concentration after dilution such that >90% of the MBP would be in the liganded form. ATP hydrolysis by the MalFGK2 transporter was measured as described (3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google Scholar). Proteoliposomes were resuspended in 20 mmKPO4, pH 6.2, 3 mm MgCl2, 10 μm sugar substrate (if added when preparing proteoliposomes) to a final concentration of 0.45 μg of protein/ml and incubated with 100 μm [γ-32P]ATP (50 mCi/mmol) at room temperature, and released Pi was determined at various time points. Strains HS3368 and NT411 were grown at 37 °C overnight in medium 63 containing 0.4% glycerol and l μg/ml thiamine. The culture was diluted 1:20 into 200 ml of the same medium and grown to a cell density of 2 × 109 cells. Membrane vesicles were prepared by a modification (20Dean D.A. Fikes J.D. Gehring K. Bassford P.J. Nikaido H. J. Bacteriol. 1989; 171: 503-510Crossref PubMed Google Scholar) of the Kaback procedure (21Kaback H.R. Methods Enzymol. 1974; 31: 698-709Crossref PubMed Scopus (124) Google Scholar), except that vesicles were separated from unlysed spheroplasts and whole cells by two centrifugations at 2000 × g for 10 min and then collected by centrifugation at 35,000 × g for 15 min. Vesicles were resuspended in 20 mm KPO4, pH 6.2, 3 mm MgCl2 to a final concentration of 1.5–2.0 mg of protein/ml, electron donors (10 mmascorbate, 100 μm phenazine methosulfate) were then added as indicated, and the preparation was used immediately. [14C]Maltose (150 μCi/μmol), [3H]maltotetraose (150 μCi/μmol), or [3H]maltotetraitol (120 μCi/μmol) was added to a final concentration of 10 μm, and 25-μl aliquots were removed at specified time points, diluted 1:10 with cold 20 mm KPO4, pH 6.2, 3 mmMgCl2, and passed through a 0.22-μm Millipore GSTF filter. Filters were then washed with 5 ml of cold 50 mmLiCl, dried, and counted by liquid scintillation using Ecolume (ICN) as scintillant. The accumulation of maltose and maltotetraose inside proteoliposomes was measured as described (13Davidson A.L. Nikaido H. J. Biol. Chem. 1990; 265: 4254-4260Abstract Full Text PDF PubMed Google Scholar). Proteoliposomes were prepared as above (see “ATP Hydrolysis in Proteoliposomes”) except that neither MBP nor substrate was added. Instead, ATP was added prior to dilution to give a final concentration of 5 mm after dilution. Proteoliposomes were resuspended to a final concentration of 0.45 μg of protein/ml in 20 mm KPO4, pH 6.2, 3 mmMgCl2 with or without 1 μm MBP and incubated with 10 μm [14C]maltose (150 μCi/μmol), 10 μm [3H]maltotetraose (150 μCi/μmol), or 10 μm [3H]maltotetraitol (120 μCi/μmol) at room temperature. At specified times, 25–40 μl of the reaction mixture was diluted 1:10 with 20 mmKPO4, pH 6.2, 3 mm MgCl2, filtered through a Millipore filter (0.22-μm GTSF), and washed with 5 ml of 50 mm LiCl. Filters were dried and counted as described above. Cells were grown in M63 medium containing 1 μg/ml thiamine, 0.4% glycerol, any necessary antibiotics, and 250 μm IPTG (if required), at 37 °C to a density between 2 × 108 and 5 × 108 cells/ml. Cells were then harvested and osmotically shocked as described above. The shock fluid containing periplasmic proteins was cleared of cells and cellular debris by passage through Whatman 1 filter paper. Proteins were then separated via SDS-polyacrylamide gel electrophoresis (2Davidson A.L Nikaido H. J. Biol. Chem. 1991; 266: 8946-8951Abstract Full Text PDF PubMed Google Scholar) and either visualized by Coomassie Blue staining or transferred to a nitrocellulose membrane for immunoblot analysis. The latter procedure was carried out by using an anti-MBP rabbit antiserum and alkaline phosphatase-conjugated anti-rabbit IgG antibody (Sigma) (22Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 505Google Scholar). The total amount of MBP loaded in each lane was quantified by comparison with serial dilutions of a pure MBP standard. Periplasmic MBP concentrations were then estimated, based on the total dry weight per cell of 2.7 × 10−12 g (23Neidhardt F.C. Umbarger H.E. Neidhardt F.C. Curtiss R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 13-16Google Scholar) and the periplasmic volume, assumed to be 30% of the total cell volume (24Stock J.B. Rauch B. Roseman S. J. Biol. Chem. 1977; 252: 7850-7861Abstract Full Text PDF PubMed Google Scholar). Strains to be used in all experiments were grown overnight at 37 °C in M63 medium (17Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 82-85Google Scholar) containing 1 μg/ml thiamine, 0.4% glycerol, and any necessary antibiotics. Overnight cultures were diluted 1:20 into fresh medium of the same composition, except for the addition of 250 μm IPTG (if required), and grown at 37 °C to a density of approximately 5 × 108 cells/ml. Cells were washed twice with M63 and then resuspended in the same medium at a density of 2 × 109 cells/ml. The assay was initiated by the addition of labeled substrate ([14C]maltose (75–150 μCi/μmol), [3H]maltotetraose (200 μCi/μmol), or [3H]maltotetraitol (200 μCi/μmol)) to a final concentration of 10 μm. Portions of 50 μl each were removed, diluted 1:10 with cold M63, and filtered through a 0.45-μm HAWP Millipore filter. Filters were then washed with 5 ml of 50 mm LiCl, dried, and counted as described above. All values were corrected for background counts on filters. To assay the ability of strains to transport and metabolize various substrates, overnight cultures were diluted 1:200 into 2.0 ml of fresh M63 media containing 1 μg/μl thiamine, any necessary antibiotics, 250 μm IPTG (if required), and maltose, maltotetraose, or maltotetraitol at the indicated concentration. Cultures were grown at 37 °C with continuous shaking, and growth was scored at various times. Either the BCA protein assay (Pierce) or the method of Brown et al. (25Brown R.E. Jarvis K.L. Hyland K.J. Anal. Biochem. 1989; 180: 136-139Crossref PubMed Scopus (576) Google Scholar) was used. The wild type complex, MalFGK2, required the presence of both maltose and MBP inside the vesicles to hydrolyze, rapidly, ATP added from the outside, as noted earlier (3Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (231) Google Scholar) (data not shown). When MBP and various ligands were added inside proteoliposomes at concentrations that would make MBP more than 95% liganded, only those ligands that bound to MBP via the R mode stimulated ATP hydrolysis by the MalFGK2complex (Table II). Interestingly, maltotriose and maltotetraose both stimulated the transporter to a slightly higher degree than did maltose and maltohexaose. In contrast, ligands that bound to MBP exclusively via the B mode, such as reduced, oxidized, or cyclic maltodextrin derivatives, caused little stimulation (Table II). A marginal stimulation seen with maltotriitol and maltotetraitol could have been caused by traces of unmodified maltodextrins remaining in these preparations.Table IIBinding of ligands to wild-type MBP and the stimulation of ATP hydrolysisLigandBinding to MBPATP hydrolysisK dBinding modeBy MalFGK2By MalFG511K21 min10 min1 min10 minμmnmol/mg proteinNone3.84.64.613Maltose1.0R15409.434Maltitol50B3.23.37.931Maltotriose0.2R22531150Malotriitol75B4.34.2ND2-aND, not determined.NDMaltotetraose1.6R27601259Maltotetraitol7.6B4.8101448Maltohexaose2.8R14418.044Maltohexaitol2.9B4.36.66.727Maltohexaonic acid3.3B1.80.99.036β-Cyclodextrin1.0B2.21.93.29.82-a ND, not determined. Open table in a new tab Transport by the wild type MalFGK2 transporter was examined in various systems. Fig. 1 A shows the accumulation of various substrates into proteoliposomes. In these experiments, ATP was trapped inside the proteoliposomes, and wild type MBP and substrate were added to the outside. Clearly, both maltose and maltotetraose (binding to MBP via the R mode) were transported quite well, but maltotetraitol (binding via the B mode) was not accumulated to a significant level. Similar results were obtained when right side-out membrane vesicles from HS3368 (containing the wild type transporter) were used; the addition of maltose, maltotetraose, and maltotetraitol (in addition to MBP) resulted in the uptake of 0.38, 0.18, and 0.02 nmol of substrate/mg of protein/min, respectively. Without MBP, the addition of these sugars produced a residual uptake value of 0.02–0.03 nmol/mg of protein/min. There were some differences between the membrane vesicles and proteoliposomes. First, the uptake rates of maltose and maltotetraose were much higher in proteoliposomes; this is most likely due to the partial purification of the transporter via selective solubilization. Second, maltotetraose was transported into proteoliposomes better than maltose, whereas in membrane vesicles the opposite was true. A possible explanation is that many of the inner membrane vesicles were surrounded by residual outer membranes, which could have hindered the access of maltotetraose. We also assayed substrate accumulation in whole cells of strain HS3368 (Fig. 1 B). Interestingly, maltose was transported approximately 25 times more rapidly than maltotetraose. Maltodextrin transport in whole cells is most likely limited by diffusion through the LamB channel (26Luckey M. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 167-171Crossref PubMed Scopus (179) Google Scholar, 27Freundlieb S. Ehmann U. Boos W. J. Biol. Chem. 1988; 263: 314-320Abstract Full Text PDF PubMed Google Scholar). Maltotetraitol accumulation was close to the base-line level. The malE254 allele was isolated by Wandersman et al. (10Wandersman C. Schwartz M. Ferenci T. J. Bacteriol. 1979; 140: 1-13Crossref PubMed Google Scholar) and is a member of a class of “maltodextrin-negative” malE mutants, which are unable to utilize maltodextrins although their MBPs have good affinity for these substrates. The MalE254 MBP, unlike wild type MBP, binds both maltodextrins and their nonreducing derivatives exclusively by the B or open mode (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, it binds maltose (at high concentrations) by the R mode (5Hall J.A. Gehring K. Nikaido H. J. Biol. Chem. 1997; 272: 17605-17609Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The MalE254 MBP was trapped inside proteoliposomes containing the wild type MalFGK2 complex together with various ligands (at concentrations that would assure at least 90% saturation of MalE254 MBP), and ATP hydrolysis was measured. We found that this mutant MBP could stimulate ATP hydrolysis only with maltose (Fig. 2). The maltotetraose-MalE254 complex showed no stimulation of ATP hydrolysis (Fig. 2), in contrast to the strong stimulatory activity of maltotetraose-wild type MBP complex (TableII). ThemalG511 allele is a member of a class of mutantmalF and malG alleles that allow a cell to transport maltose in the a" @default.
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• W1968204553 title "Two Modes of Ligand Binding in Maltose-binding Protein ofEscherichia coli" @default.
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