Biomedical subjects

H R Kaback

Publications and source records attributed to H R Kaback.

At least 91 records · Page 5Linked to original sources

PubMed1996

Monoclonal antibody 4B1 alters the pKa of a carboxylic acid at position 325 (helix X) of the lactose permease of Escherichia coli.

A carboxylic acid at position 325 in helix X is obligatory for lactose/H+ symport at a step corresponding to deprotonation of lactose permease [Carrasco, N. et al. (1989) Biochemistry 28, 2533-2539]. In this paper, pH profiles for active transport, efflux, and equilibrium exchange are analyzed for wild-type permease and mutant Glu325-->Asp. With respect to active transport and efflux down a concentration gradient, both of which involve net H+ translocation and are defective in the mutant, the wild-type and the mutant exhibit similar profiles, and at no pH is the mutant stimulated relative to the wild-type. Strikingly, exchange which does not involve H+ translocation is comparable in the wild-type and the Glu325-->Asp mutant below pH 7.5. Above pH 7.5, however, the exchange activity of the mutant is progressively and reversibly inhibited with a midpoint at about pH 8.5; while the exchange activity of wild-type permease is only mildly decreased above pH 9.5, and exchange by Glu325-->Ala or Glu325-->Gln permease is comparable to wild-type and unaffected by pH. The findings are consistent with the idea that translocation of the ternary complex between the permease, lactose, and H+ does not tolerate a negative charge at position 325. In wild-type permease, the electrostatic interaction between Glu325 (helix X) and Arg302 (helix IX) is sufficiently strong that the carboxylate is unaffected by pH. In contrast, with Asp at position 325, the electrostatic interaction is broken, the carboxylate becomes protonated, and the acid exhibits a pKa of about 8.5. Monoclonal antibody 4B1 binds to the periplasmic loop between helices VII and VIII of the permease [Sun, J. et al. (1996) Biochemistry 35, 990-998] and mimics the Glu325 mutants. Dramatically, 4B1 shifts the apparent pKa for exchange from about pH 8.5 to 7.5 in the Glu325-->Asp mutant with little or no effect on the wild-type or the Glu325-->Ala mutant. The findings are consistent with the conclusion that the uncoupling effect of 4B1 involves a conformational change in helix VII and/or VIII that secondarily alters the pKa of the essential carboxylic acid at position 325.

Amino Acid Sequence↗

PubMed1996

A phospholipid acts as a chaperone in assembly of a membrane transport protein.

A mutant of Escherichia coli lacking phosphatidylethanolamine (PE) and a monoclonal antibody (mAb 4B1) directed against a conformationally sensitive epitope (4B1) of lactose permease were used to establish a novel role for a phospholipid in the assembly of a membrane protein. Epitope 4B1 is readily detectable in spheroplasts and right-side-out membrane vesicles from PE-containing but not from PE-deficient cells expressing lactose permease. Lactose permease from PE-containing membranes, but not from PE-deficient membranes, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis is also recognized by mAb 4B1. If total E. coli phospholipids or PE (but not phosphatidylcholine, phosphatidylglycerol, or cardiolipin) are blotted on nitrocellulose sheets (Eastern blot) prior to transfer of proteins from SDS-polyacrylamide gels, the permease from PE-deficient cells regains its recognition by mAb 4B1. Therefore, PE is required during assembly to form epitope 4B1, but, once formed, sufficient "conformational memory" is retained in the permease to either retain or reform this epitope in the absence of PE. Lactose permease lacking epitope 4B1 can be induced to form the epitope if partially denatured and then renatured in the presence of PE specifically. These results establish for the first time a role for PE as a molecular chaperone in the assembly of the lactose permease.

Antibodies, Monoclonal↗

PubMed1996

Cysteine-scanning mutagenesis of helix VI and the flanking hydrophilic domains on the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in putative transmembrane helix VI and the flanking hydrophilic loops (residues 164- 211) was replaced individually with Cys. Of the 48 mutants, 43 accumulate lactose at highly significant rates to > 80% of the steady state observed with C-less permease. Three mutants (Phe185--> Cys, Ala187--> Cys, and Phe208--> Cys) exhibit lower but significant levels of accumulation (30-60% of C-less). Cys replacement for Ala177 or Leu184 results in low transport activity (ca. 20%) in the C-less background but much higher activity (60-70%) in the wild type. Immunoblot analysis reveals that all of the mutants are inserted into the membrane at concentrations comparable to that of C-less perrmease. The transport activity of the great majority of the mutants is unaffected by treatment with N-ethylmaleimide (NEM). Relatively modest but significant inactivation (ca. 50%) is observed with mutants Phe170--> Cys, Gly173--> Cys, and Ala187--> Cys, and these positions cluster on the same face of the helix VI. Moreover, the two positions where single Cys replacements result in low activity (Ala177 and Lcu184) are on the same face of helix VI. The results demonstrate the following. (i) Permease function is not disrupted by replacement of most residues with Cys, but function is disrupted when some of the residues are further altered by addition of the NeM moiety. (ii) The latter residues lie on a stripe down one face of an alpha-helix, and within the same stripe are residues where Cys substitution itself leads ti inhibition of function.

Amino Acid Sequence↗

PubMed1996

Purification and functional characterization of the C-terminal half of the lactose permease of Escherichia coli.

The lactose permease has been expressed in contiguous, non-overlapping polypeptide fragments containing the N-terminal (N6) and C-terminal (C6) transmembrane domains of the protein [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325; Zen, K., et al. (1994) Biochemistry 33, 8198]. When expressed individually, N6 and C6 are unstable and do not catalyze active transport. However, when expressed simultaneously, the polypeptides stabilize each other and form a complex that catalyzes active lactose transport. Moreover, a deletion construct containing the first transmembrane domain and the six C-terminal transmembrane domains mediates downhill lactose translocation [Bibi et al. (1991) proc. Natl. Acad. Sci. U.S.A. 88, 7271]. Here we report that C6 can be expressed independently in a relatively stable form that binds monoclonal antibodies 4B1 and 4B11, which interact with conformationally dependent epitopes on the periplasmic and cytoplasmic surfaces of the membrane, respectively. In addition, C6 retains the ability to catalyze lactose translocation down a concentration gradient in a specific manner. Finally, as observed with full-length Val331Cys permease, beta-D-galactopyranosyl 10thio-beta-D-galactopyranoside quenches the fluorescence of 2-(4'-maleimidylanilino)naphthalene- 6-sulfonic acid (MIANS)- labeled C6 with a single-Cys residue in place of Val331, exhibiting as apparent Kd of 0.2 mM. Unlike full-length Val331Cys permease, however, ligand does not induce a chance in the position of the emission maximum of MIANS-labeled C6(Val331Cys) permease not in the reactivity of C6 (Val331Cys) permease with MINAS. the results indicate that C6 retains a conformation similar to that on the native permease and that most of the structure required of high-affinity binding and substrate translocation is located in the C-terminal half of the molecule.

Amino Acid Sequence↗

PubMed1996

Probing the conformation of the lactose permease of Escherichia coli by in situ site-directed sulfhydryl modification.

By using site-directed chemical labeling of lactose permease, conformational changes induced by ligand binding are observed in the native membrane of Escherichia coli. Membranes containing permease mutants with a single-Cys residue and a biotin-acceptor domain were labeled with radioactive N-ethylmaleimide (NEM) in the presence or absence of beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or a proton electrochemical gradient, followed by solubilization in n-dodecyl beta-D-maltopyranoside and adsorption to avidin. TDG-induced enhancement of the reactivity of membrane-embedded Val315-->Cys (helix X) permease is observed, while the reactivity of Val331-->Cys (helix X) permease is inhibited by ligand binding or imposition of a proton electrochemical gradient. In contrast, the reactivity of permease with a single native Cys residue at position 148 (helix V) is blocked by TDG, but unaffected by the proton electrochemical gradient. Furthermore, as shown with right-side-out and inside-out membrane vesicles, the accessibility of Cys148 to either NEM or impermeant methanethiosulfonate derivatives is comparable from both sides of the membrane. On the other hand, TDG protects Cys148 from alkylation more effectively in right-side-out vesicles (apparent KD = 20-50 microM) than inside-out vesicles (apparent KD ca. 1.0 microM). The findings provide strong support for the conclusion that the permease retains close to native conformation in n-dodecyl-beta-D-maltopyranoside. In addition, the results are consistent with the idea that lactose permease has two binding sites: one with higher affinity on the periplasmic surface of the membrane and another with lower affinity on the cytoplasmic surface.

Amino Acid Sequence↗

PubMed1996

Membrane topology of the melibiose permease of Escherichia coli studied by melB-phoA fusion analysis.

In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assayed for alkaline phosphatase activity. In general agreement with a previously suggested secondary structure model of melibiose permease [Botfield, M. C., Naguchi, K., Tsuchiya, T., & Wilson, T.H. (1992) J. Biol. Chem. 267, 1818], clusters of fusions exhibiting low and high phosphatase activity fusions alternate along the primary sequence. Fusions with high activity generally cluster at residues predicted to be in the periplasmic half of transmembrane domains or in periplasmic loops, while fusions with low activity cluster at residues predicted to be in the cytoplasmic half of transmembrane domains or in cytoplasmic loops. Taken together, the findings strongly support the contention that melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane with the N and C termini on the cytoplasmic face of the membrane. Moreover, on the basis of the finding that the cytoplasmic half of an out-going segment is sufficient for alkaline phosphatase export to the periplasm while the periplasmic half of an in-going segment prevents it [Calamia, T., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937], the activity profile of the melibiose permease-alkaline phosphatase fusions is consistent with the predicted topology of seven of 12 transmembrane segments. However, five transmembrane domains require adjustment, and as a consequence, the size of the central cytoplasmic loop is reduced and a significant number of charged residues are shifted from a hydrophilic to a hydrophobic domain in this region of the transporter.

Alkaline Phosphatase↗

PubMed1996

Association between the amino- and carboxyl-terminal halves of lactose permease is specific and mediated by multiple transmembrane domains.

Lactose permease of Escherichia coli is a polytopic membrane transport protein containing 12 membrane-spanning segments. When the amino (N6)- and carboxy (C6)-terminal halves are expressed as separate gene fragments, association of the first half (N6) of the permease with the second half (C6) is necessary for stable insertion of C6 [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325-4329]. In this report we demonstrate that N6-C6 interaction is specific, since N6 fragments derived from the structurally related tetracycline or sucrose transporters are unable to stabilize insertion of C6 from lactose permease. Furthermore, this association appears to be mediated by multiple transmembrane domains, since co-expression of progressively truncated N-terminal fragments (N5, N4, N3, N2, N1) with C6 leads to markedly decreased, but detectable amounts of C6 in the membrane. The results indicate that the N- and C-terminal six transmembrane domains of lactose permease are integrated into the membrane as separate units, and insertion of the C-terminal half is directed by specific interactions with the N-terminal half of the protein.

Amino Acid Sequence↗

PubMed1996

Identification of the epitope for monoclonal antibody 4B1 which uncouples lactose and proton translocation in the lactose permease of Escherichia coli.

Monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and H+ translocation in a manner indicating that it blocks deprotonation [Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H. R. (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., Carrasco, N., & Kaback, H. R. (1984) Biochemistry 23, 3688]. In this paper, 4B1 binding to purified lactose permease is shown to exhibit a KD of about 5 x 10(-10) M by surface plasmon resonance. Furthermore, the combined use of mutants containing 6 contiguous His residues in each periplasmic loop in the permease and Cys-scanning mutagenesis in conjunction with chemical labeling demonstrates that 4B1 binds specifically to the periplasmic loop between helices VII and VIII and that Phe247 and Gly254 are the primary determinants. Remarkably, although 4B1 binding uncouples lactose and H+ translocation, none of the amino acid residues in periplasmic loops, particularly Phe247 or Gly254, play an important role in the transport mechanism. Moreover, binding of avidin to biotinylated Glu255-->Cys in the loop containing the epitope has no effect on transport activity. Therefore, the uncoupling effect of 4B1 involves highly specific interactions which in all likelihood exert a torsional effect on the loop, resulting in a conformational change in helix VII and/or VIII that alters the pKas of residues involved in lactose-coupled H+ translocation.

Amino Acid Sequence↗

PubMed1996

Engineering the lac permease for purification and crystallization.

The lactose permease is being used as a model system for the rational redesign of a membrane protein with the goal of increasing the likelihood of crystallization. Various modifications to the protein have been added for the purposes of purification, stability, and potential for crystallization. The addition of six consecutive histidines at the C-terminus of the protein allows for rapid purification by nickel-chelate chromatography, and the insertion of an entire protein domain into one of the inner cytoplasmic loops of the permease gives the resulting protein a larger hydrophilic surface area. The increase in polar surface area makes the fusion protein easier to handle and more likely to crystallize. In particular, the introduction of cytochrome b562 of E. coli into the central hydrophilic domain the lac permease results in a fusion protein with the transport activity of the permease and the visible absorbance spectrum of the cytochrome. The "red permease" is very easy to monitor through the steps of expression, purification, concentration, and crystallization.

Avidin↗

PubMed1995

A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: evaluation with T4 lysozyme.

The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate intramolecular distances in a wide variety of proteins. In this report, a His-Xaa3-His metal ion binding motif was introduced at the N terminus of the long interdomain helix of T4 lysozyme (Lys-65 --> His/Gln-69 --> His) of three mutants, each containing a single nitroxide-labeled cysteine residue at position 71, 76, or 80. The results show that Cu(II)-induced relaxation effects on the nitroxide can be quantitatively analyzed in terms of interspin distance in the range of 10-25 A using Redfield theory, as first suggested by Leigh [Leigh, J.S. (1970) J. Chem. Phys. 52, 2608-2612]. Of particular interest is the observation that distances can be determined both under rigid lattice conditions in frozen solution and in the presence of motion of the spins at room temperature under physiological conditions. The method should be particularly attractive for investigating structure in membrane proteins that are difficult to crystallize. In the accompanying paper, the technique is applied to a polytopic membrane protein, lactose permease.

Bacteriophage T4↗

PubMed1995

Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.

Amino Acid Sequence↗

PubMed1995

Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli. 1. Proximity of helix VII (Asp237 and Asp240) with helices X (Lys319) and XI (Lys358).

The lactose permease of Escherichia coli contains two pairs of oppositely charged residues that interact functionally, Asp240 (helix VII)/Lys319 (helix X) and Asp237 (helix VII)/Lys358 (helix XI). Single- and double-His replacement mutants at these positions have been constructed and characterized with respect to transport activity and Mn2+ binding. The following results confirm the functional interactions between both sets of residues: (i) At pH 7.5, where the imidazole is likely to be unprotonated, the double-His mutants Asp237 --> His/Lys358 --> His and Asp240 --> His/Lys319 --> His exhibit significant transport activity while the single-His mutants Lys319 --> His and Lys358 --> His are inactive. (ii) At pH 5.5, where the imidazole is likely to be protonated, the double-His mutants Asp240 --> His/Lys319 --> His and Asp237 --> His/Lys358 --> His are inactive; however, the single-His mutant Lys319 --> His exhibits significant activity. (iii) The single-His mutant Asp237 --> His or ASP240 --> His is inactive at all pH values tested. In addition, a pH titration of Asp237 --> His/Lys358 --> His permease activity exhibits a midpoint at about 6.2. Finally, the purified mutant proteins Asp237 --> His/Lys358 --> His and Asp240 --> His/Lys319 --> His were assayed for Mn2+ binding by electron paramagnetic resonance spectroscopy. Asp237 --> His/Lys358 --> His permease binds Mn2+ with a stoichiometry of unity at pH 7.5, but much less binding is observed at pH 5.5, demonstrating directly that helix VII (Asp237) is in close proximity to helix XI (Lys358).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Use of designed metal-binding sites to study helix proximity in the lactose permease of Escherichia coli. 2. Proximity of helix IX (Arg302) with helix X (His322 and Glu325).

Engineering divalent metal-binding sites into the lactose permease of Escherichia coli by introducing bis-His residues has been utilized to confirm the proximity of helices VIII (Glu269 --> His) and X (His322) [Jung, K., Voss, J., He, M., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 6272] and helices VII (Asp237 --> His) and XI (Lys358 --> His) [He, M. M., Voss, J., Hubbell, W. L., & Kaback, H.R. (1995) Biochemistry 34, 00000--00000]. In this paper, the approach is used to confirm and extend the relationship between helices IX (Arg302) and X (His322 and Glu325) [Jung, K., Jung, H., Wu, J., Prive, G. G., l& Kaback, H. R. (1993) Biochemistry 32, 12273]. Thus, mutants Arg302 --> His, Glu325 --> His, and Arg302 --> His/Glu325 --> His were constructed, and Mn2+ binding was assayed by electron paramagnetic resonance. Mutant Arg302 --> His binds Mn2+ with a KD of about 24 microM and a stoichiometry approximating unity in all likelihood because the His residue at position 302 forms a metal-binding site in conjunction with the native His residue at position 322. Mutant Arg302 --> His/Glu325 --> His also binds Mn2+ with a 1:1 stoichiometry, but the KD is decreased to about 13 microM. The results suggest that Arg302 is sufficiently close to both Glu325 and His322 to form a tridentate metal-binding site in mutant Arg302 --> His/Glu325 --> His. In contrast, replacement of Glu325 with His in permease with a native His residue at position 322 does not lead to Mn2+ binding. The results provide strong support for the helix packing model proposed.

Amino Acid Sequence↗

PubMed1995

Membrane topology of helices VII and XI in the lactose permease of Escherichia coli studied by lacY-phoA fusion analysis and site-directed spectroscopy.

The use of lactose permease-alkaline phosphatase fusions (lacY-phoA) demonstrates that the lactose permease of Escherichia coli contains 12 transmembrane domains and that approximately half of a transmembrane domain is required to translocate alkaline phosphatase to the periplasmic surface of the membrane [Calamia, J., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937-4941]. We have now used fusion analysis in combination with site-directed spectroscopy to examine more precisely the topology of putative helices VII and XI which contain the interacting residues Asp237 and Lys358, respectively. For this purpose, alkaline phosphatase was fused to alternate amino acid residues in transmembrane domains VII and XI. A sharp increase in alkaline phosphatase activity is observed as the fusion junction proceeds from Try228 to Ile230 in helix VII and from Phe354 to Phe356 in helix XI, suggesting that these residues approximate the middle of the corresponding transmembrane helices. Analysis of fluorescence quenching of the pyrene-labeled single-Cys mutants Asp237 --> Cys or Lys358 --> Cys, as well as measurement of collision frequencies between freely diffusing paramagnetic probes and a nitroxide spin-label at these sites, also indicates that Asp237 and also Asp240, which interacts with Lys319 (helix X), are located in transmembrane domains. However, Asp237 and Asp240 are accessible both from the aqueous phase and from within the membrane. The results provide more direct evidence that the three residues are located within transmembrane helices and suggest that Asp237 and Asp240 are either located near the periplasmic surface of the membrane or exposed within a solvent-filled cleft in the permease.

Alkaline Phosphatase↗

PubMed1995

Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.

Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and derivatized with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper [OP(Cu)]. Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease in dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximately 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody. Remarkably, the same cleavage products are observed with permease embedded in the native membrane. Comparison with the C-terminal half of the permease expressed independently as a standard indicates that the 19-kDa product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI. Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mutagenesis studies. Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximately 19 kDa and at 15-16 kDa. The larger fragment probably reflects cleavage at a site near the cytoplasmic end of helix VII, whereas the 15- to 16-kDa fragment is consistent with cleavage near the cytoplasmic end of helix VIII. When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys permease), a single product is observed at 19 kDa, suggesting fragmentation at the cytoplasmic end of helix VII. However, when the reagent is moved 100 degrees in the other direction to position 147 (Gly-147 --> Cys permease), cleavage is not observed. The results suggest that helix V is in close proximity to helices VII and VIII with position 148 in the interface between the helices, position 149 facing helix VII, and position 147 facing the lipid bilayer.

Amino Acid Sequence↗

PubMed1995

Helix packing in the sucrose permease of Escherichia coli: properties of engineered charge pairs between helices VII and XI.

Of four putative intramembrane charge pairs in lactose permease, only three are conserved in the homologous sucrose permease of Escherichia coli [Bockmann, J., Heuel, H., & Lengeler, J. W. (1992) Mol. Gen. Genet. 235, 22-32]. The missing charge pair was introduced into wild-type sucrose permease by site-directed mutagenesis of Asn234 (helix VII) and Ser356 (helix XI). Individual replacement of either residue with a charged amino acid abolishes active sucrose transport with the exception of the Asn234-->Asp mutant. However, simultaneous replacement of Asn234 with Asp or Glu and Ser356 with Arg or Lys results in high activity. Thus, an acidic residue at position 234 rescues the activity of the Ser356-->Arg or Ser356-->Lys mutant, and a basic residue at position 356 rescues the activity of the Asn234-->Glu mutant. Furthermore, when expressed at a relatively low rate, the double mutant Asn234-->Asp/Ser356-->Arg is present in the membrane in a significantly greater amount than wild-type, suggesting that the charge pair improves insertion of sucrose permease into the membrane. The results indicate that helices VII and XI of sucrose permease are in close proximity and that a charge pair interaction can be established between residues 234 (helix VII) and 356 (helix XI). However, interchange of the acidic residue at position 234 with the basic residue at position 356 abolishes sucrose transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Cysteine scanning mutagenesis of helix V in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys (C-less permease), each amino acid residue in putative transmembrane helix V was replaced individually with Cys (from Met145 to Thr163). Of the 19 mutants, 13 are highly functional (60-125% of C-less permease activity), and 4 exhibit lower but significant lactose accumulation (15-45% of C-less permease). Cys replacement of Gly147 or Trp151 essentially inactivates the permease (< 10% of C-less); however, previous studies [Menezes, M. E., Roepe, P. D., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1638; Jung, K., Jung, H., et al. (1995) Biochemistry 34, 1030] demonstrate that neither of these residues is important for activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to C-less permease with the exception of Trp151-->Cys and single Cys154 permeases which are present in reduced amounts. Finally, only three of the single-Cys mutants are inactivated significantly by N-ethylmaleimide (Met145-->Cys, native Cys148, and Gly159-->Cys), and the positions of the three mutants fall on the same face of helix V.

Amino Acid Sequence↗

PubMed1995

Dynamics of lactose permease of Escherichia coli determined by site-directed chemical labeling and fluorescence spectroscopy.

Mutants with a single Cys residue in place of Phe27, Pro28, Phe29, Phe30, or Pro31 at the periplasmic end of putative transmembrane helix I were used to study the interaction of lactose permease with ligand by site-directed chemical modification or fluorescence spectroscopy. With permease embedded in the native membrane, mutant Phe27-->Cys or Phe28-->Cys is readily labeled with [14C]-N-ethylmaleimide (NEM), while mutant Phe29-->Cys, Phe30-->Cys, or Phe31-->Cys reacts less effectively. beta,D-Galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) has little or no effect on the reactivity of Phe27-->Cys, Phe29-->Cys, or Phe30-->Cys permease. Remarkably, however, Pro31-->Cys permease which is essentially unreactive in the absence of ligand becomes highly reactive in the presence of TDG. Ligand also enhances the NEM reactivity of the mutant with Cys in place of Pro28 which is presumably on the same face of helix I as position 31. The five single-Cys mutants which also contain a biotin acceptor domain in the middle cytoplasmic loop were purified by monomeric avidin-affinity chromatography in dodecyl beta,D-maltoside and subjected to site-directed fluorescence spectroscopy. Mutants Phe27-->Cys, Phe29-->Cys, and Phe30-->Cys react rapidly with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS), and reactivity is not altered in the presence of TDG. In striking contrast, mutants Pro28-->Cys and Pro31-->Cys react extremely slowly with MIANS in the absent of ligand, and TDG dramatically enhances reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗