Biomedical subjects

H R Kaback

Publications and source records attributed to H R Kaback.

At least 37 records · Page 2Linked to original sources

PubMed2000

Thiol cross-linking of transmembrane domains IV and V in the lactose permease of Escherichia coli.

Glu126 (helix IV) and Arg144 (helix V) in the lactose permease of Escherichia coli are critical for substrate binding and transport, and the two residues are in close proximity and charge-paired. By using a functional permease construct with two tandem factor Xa protease sites in the cytoplasmic loop between helices IV and V, it is shown here that Cys residues in place of Glu126 and Arg144, as well as Ala122 and Val149, spontaneously form disulfide bonds in situ, indicating that this region of transmembrane domains IV and V is in the alpha-helical conformation. To determine if the local structure or environment is perturbed by the presence of an unpaired charge, either Glu126 or Arg144 or both were replaced with Ala, and cross-linking between the Cys pair Ala122-->Cys/Val149-->Cys was studied. Ala replacement for Arg144 causes a marked decrease in cross-linking, while Ala replacement for Glu126 alone or for both Glu126 and Arg144 has little effect. The data provide strong support for the argument that Glu126 and Arg144 are within close proximity and suggest that an unpaired carboxylate at position 126 causes a structural change at the interface between helices IV and V.

Amino Acid Sequence↗

PubMed2000

Ligand recognition by the lactose permease of Escherichia coli: specificity and affinity are defined by distinct structural elements of galactopyranosides.

Specificity of substrate recognition in lactose permease is directed toward the galactosyl moiety of lactose. In this study, binding of 31 structural analogues of D-galactose was examined by site-directed N-[(14)C]ethylmaleimide-labeling of the substrate-protectable Cys148 in the binding site. Alkylation of Cys148 is blocked by D-galactose with an apparent affinity of approximately 30 mM. Epimers of D-galactose at C-3 (D-gulose) and C-4 (D-glucose) or deoxy derivatives at these positions exhibit no binding whatsoever, indicating that these OH groups participate in essential interactions. Interestingly, the C-2 epimer alpha-D-talose binds almost as well as D-galactose, while 2-deoxy-D-galactose affords no substrate protection, indicating that nonstereospecific H-bonding at C-2 is required for stable binding. No substrate protection is detected with D-fucose, L-arabinose, 6-deoxy-6-fluoro-D-galactose, 6-O-methyl-D-galactose, or D-galacturonic acid, suggesting that the C-6 OH is an essential H-bond donor. Both alpha- and beta-methyl D-galactopyranosides bind more strongly than galactose, supporting the notion that the cyclic pyranose conformation is the bound form and that the anomeric configuration at C-1 does not contribute to substrate specificity. However, methyl or allyl alpha-D-galactopyranosides exhibit 60-fold lower apparent K(d)'s than D-galactose, demonstrating that binding affinity is significantly influenced by the functional group at C-1 and its orientation. Taken together, the observations confirm and extend the current binding site model [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] and indicate that specificity toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by hydrophobic interactions with the nongalactosyl moiety.

Bacterial Proteins↗

PubMed2000

Proteomics on full-length membrane proteins using mass spectrometry.

A general technique has been developed that allows rapid mass spectrometric analysis of full-length membrane proteins [Whitelegge, J. P., le Coutre, J., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10695-10698]. Using in-line HPLC electrospray ionization mass spectrometry (LC-MS), different native and recombinant bacterial membrane proteins of up to 61 kDa are characterized. Mass spectrometric data of four entirely different membrane proteins from three bacterial organisms, two transporters, a channel, and a porin protein are presented. In addition to determination of the molecular mass with an accuracy of +/-0.01%, the technique monitors alkylation or oxidation of single Cys residues and errors in deduced amino acid sequences. Finally, using in-line LC-MS, unknown proteins can be identified from solubilized Escherichia coli membranes without prior purification.

Alkylation↗

PubMed2000

Thiol cross-linking of cytoplasmic loops in the lactose permease of Escherichia coli.

The N- and C-terminal halves of lactose permease, each with a single-Cys residue in a cytoplasmic loop, were coexpressed, and cross-linking was studied in the absence or presence of ligand. Out of the 68 paired-Cys mutants in cytoplasmic loops IV/V and VIII/IX or X/XI, three pairs in loop IV/V and X/XI, (i) Arg135 --> Cys/Thr338 --> Cys, (ii) Arg134 --> Cys/Val343 --> Cys, and (iii) Arg134 --> Cys/Phe345 --> Cys, form a spontaneous disulfide bond, indicating that loops IV/V and X/XI are in close proximity. In addition, specific paired-Cys residues in loop IV/V (132-138) and loop VIII/IX (282-290) or loop X/XI (335-345) cross-link with iodine and/or the homobifunctional cross-linking agents N, N'-o-phenylenedimaleimide, N,N'-p-phenylenedimaleimide, and 1, 6-bis(maleimido)hexane. The results demonstrate that loop IV/V is close to both loop VIII/IX and loop X/XI. On the other hand, similar though less extensive cross-linking studies indicate that neither the N terminus nor loop II/III appear to be close to loops VIII/IX or X/XI. The findings suggest that the longer cytoplasmic loops are highly flexible and interact in a largely random fashion. However, although a Cys residue at position 134 in loop IV/V, for example, is able to cross-link with a Cys residue at each position in loop VIII/IX or loop X/XI, Cys residues at other positions in loop IV/V exhibit markedly different cross-linking patterns. Therefore, although the domains appear to be very flexible, the interactions are not completely random, suggesting that there are probably at least some structural constraints that limit the degree of flexibility. In addition, evidence is presented suggesting that ligand binding induces conformational alterations between loop IV/V and loop VIII/IX or X/XI.

Binding Sites↗

PubMed1999

Location of helix III in the lactose permease of Escherichia coli as determined by site-directed thiol cross-linking.

The six N-terminal transmembrane helices (N(6)) and the six C-terminal transmembrane helices (C(6)) in the lactose permease of Escherichia coli, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices III (position 78, 81, 84, 86, 87, 88, 90, 93, or 96) and VII (position 227, 228, 231, 232, 235, 238, 239, 241, 243, 245, or 246) was examined by using iodine or two rigid homobifunctional thiol-specific cross-linking reagents with different lengths [N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N, N'-p-phenylenedimaleimide (p-PDM; 10 A)]. Cys residues in the periplasmic half of helix III (position 87, 93, or 96) cross-link to Cys residues in the periplasmic half of helix VII (position 235, 238, 239, 241, or 245). In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices III (position 78 or 81) and VII (position 227, 228, 213, 232, or 235). Therefore, the periplasmic halves of helices III and VII are in close proximity, and the helices tilt away from each other toward the cytoplasmic face of the membrane. On the basis of the findings, a modified helix packing model for the permease is presented.

Biological Transport, Active↗

PubMed1999

Nitroxide scanning electron paramagnetic resonance of helices IV and V and the intervening loop in the lactose permease of Escherichia coli.

Glu126 and Arg144 in helices IV and V, respectively, in the lactose permease of Escherichia coli, which play an indispensable role in substrate binding, are charge-paired and in close proximity [Venkatesan, P., Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807; Zhao, M., Zen, K.-C., et al. (1999) Biochemistry 38, 7407-7412]. Since hydropathy plots indicate that these residues are at the membrane-water interface at the cytoplasmic surface of the membrane, site-directed nitroxide scanning electron paramagnetic resonance (EPR) has been carried out on this region of the permease. Thirty-one single-Cys permease mutants were spin-labeled and examined by conventional and power saturation EPR. The motional freedom of the side chains, as well as accessibility to O(2) or potassium chromium oxalate (CrOx), indicates that the loop between helices IV and V (loop IV/V) is considerably smaller than predicted by hydropathy plots, extending only from about Val132 to Phe138 and that Glu126 and Arg144 are probably within the membrane. Although ligand binding has no effect on the mobility of the labeled side chains, a marked increase in CrOx and O(2) accessibility is observed at position 137, as well as significant changes in accessibility to CrOx on one face of helix V. It is concluded that ligand binding induces a conformational change in the vicinity of the binding site, resulting in increased accessibility of position 137 in loop IV/V to solvent.

Amino Acid Sequence↗

PubMed1999

Conformational flexibility at the substrate binding site in the lactose permease of Escherichia coli.

Glu-126 (helix IV) and Arg-144 (helix V) are charge paired and play a critical role in substrate binding in the lactose permease of Escherichia coli. When Glu-126 is replaced with Asp, the permease has relatively high activity, implying that helix V has sufficient flexibility to allow Arg-144 to accommodate the decreased length of the carboxylate-containing side chain of Asp-126. Helices IV and V contain five Gly residues at positions 115, 121, 141, 147, and 150, all of which are conserved in the oligosaccharide/H(+) symport family of the major facilitator superfamily. To test the notion that these residues may contribute to conformational flexibility, each residue was replaced with Ala in either the wild type or the Glu-126 --> Asp mutant. Although the replacements are well tolerated in the wild type, the mutations severely inactivate substrate binding and transport in the Glu-126 --> Asp background, with the exception of Gly-121 --> Ala, which retains significant activity. Strikingly, moreover, in two instances (Gly-150 --> Ala and Gly-141 --> Ala), significant activity is recovered when Ala residues at approximately parallel positions in helix IV (Ala-122 or Ala-127, respectively) are replaced with Gly. In addition to providing further evidence that the major determinants for substrate binding in the permease are at the interface between helices IV and V, the findings indicate that the region is conformationally flexible.

Binding Sites↗

PubMed1999

Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins.

Genes encoding membrane proteins comprise a substantial proportion of genomes sequenced to date, but ability to perform structural studies on this portion of the proteome is limited. Electrospray ionization-MS (ESI-MS) of an intact protein generates a profile defining the native covalent state of the gene product and its heterogeneity. Here we apply ESI-MS technology with accuracy exceeding 0.01% to a hydrophobic membrane protein with 12-transmembrane alpha-helices, the full-length lactose permease from Escherichia coli. Furthermore, ESI-MS is used to titrate reactive thiols with N-ethylmaleimide. Treatment of the native protein solubilized in detergent micelles reveals only two reactive thiols, and both are protected by a substrate analog.

Chromatography, Gel↗

PubMed1999

Proximity relationships between helices I and XI or XII in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking.

The lactose permease of Escherichia coli was expressed in two fragments (split permease), each with a Cys residue, and cross-linking was studied. Split permease with a discontinuity in either loop II/III (N2C10permease) or loop VI/VII (N6C6permease) was used. Proximity of multiple pairs of Cys residues in helices I and XI or XII was examined by using three homobifunctional thiol-specific cross-linking reagents of different lengths and flexibilities (6 A, rigid; 10 A, rigid; 16 A, flexible) or iodine. Cys residues in the periplasmic half of helix I cross-link to Cys residues in the periplasmic half of helix XI. In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices I and XI. Therefore, the periplasmic halves of helices I and XI are in close proximity, and the helices tilt away from each other towards the cytoplasmic face of the membrane. Cross-linking is also found with paired Cys residues near the middle of helices I and XII, but not with paired Cys residues near either end of the helices. Thus, helices I and XII are in close proximity only in the approximate middle of the membrane. Based on the findings, a modified helix packing model is proposed.

Biological Transport, Active↗

PubMed1999

Estimating loop-helix interfaces in a polytopic membrane protein by deletion analysis.

Insertions of amino acids into transmembrane helices of polytopic membrane proteins disrupt helix-helix interactions with loss of function, while insertions into loops have little effect on transmembrane helices and therefore little effect on activity [Braun, P., Persson, B., Kaback, H. R., and von Heijne, G. (1997) J. Biol. Chem. 272, 29566-29571]. Here the inverse approach, amino acid deletion, is utilized systematically to approximate loop-helix boundaries in the lactose permease of Escherichia coli. Starting with deletion mutants in the periplasmic loop between helices VII and VIII (loop VII/VIII), which has been defined by immunological analysis and nitroxide-scanning electron paramagnetic resonance spectroscopy, it is shown that mutants with single or multiple deletions in the central portion of the loop retain significant transport activity, while deletion of amino acid residues near the loop-helix boundaries or within the flanking helices leads to complete inactivation. Results consistent with hydropathy analysis are obtained with loops VI/VII, VIII/IX, and IX/X and the flanking helices. In contrast, deletion analysis of loops III/IV, IV/V, and V/VI and the flanking helices indicates that this region of the permease differs from hydropathy predictions. More specifically, evidence is presented supporting the contention that Glu126 and Arg144 which are charge paired and critical for substrate binding are within helices IV and V, respectively.

Amino Acid Sequence↗

PubMed1999

Proximity between Glu126 and Arg144 in the lactose permease of Escherichia coli.

Evidence has been presented [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] that Glu126 (helix IV) and Arg144 (helix V) which are critical for substrate binding in the lactose permease of Escherichia coli are charge paired and therefore in close proximity. To test this conclusion more directly, three different site-directed spectroscopic techniques were applied to permease mutants in which Glu126 and/or Arg144 were replaced with either His or Cys residues. (1) Glu126-->His/Arg144-->His permease containing a biotin acceptor domain was purified by monomeric avidin affinity chromatography, and Mn(II) binding was assessed by electron paramagnetic resonance spectroscopy. The mutant protein binds Mn(II) with a KD of about 40 microM at pH 7.5, while no binding is observed at pH 5.5. In addition, no binding is detected with Glu126-->His or Arg144-->His permease. (2) Permease with Glu126-->Cys/Arg144-->Cys and a biotin acceptor domain was purified, labeled with a thiol-specific nitroxide spin-label, and shown to exhibit spin-spin interactions in the frozen state after reconstitution into proteoliposomes. (3) Glu126-->Cys/Arg144-->Cys permease with a biotin acceptor domain was purified and labeled with a thiol-specific pyrene derivative, and fluorescence spectra were obtained after reconstitution into lipid bilayers. An excimer band is observed with the reconstituted E126C/R144C mutant, but not with either single-Cys mutant or when the single-Cys mutants are mixed prior to reconstitution. The results provide strong support for the conclusion that Glu126 (helix IV) and Arg144 (helix V) are in close physical proximity.

Amino Acid Sequence↗

PubMed1999

Deduction of consensus binding sequences on proteins that bind IIAGlc of the phosphoenolpyruvate:sugar phosphotransferase system by cysteine scanning mutagenesis of Escherichia coli lactose permease.

Mediated by the protein IIAGlc, the phosphoenolpyruvate:sugar phosphotransferase system plays a role in the regulation of activity of other sugar transport systems in Escherichia coli. By using a direct binding assay, a collection of single-Cys replacement mutants in cytoplasmic loops of lactose permease were evaluated for their capacity to bind IIAGlc. Selected Cys replacements in loops IV/V or VI/VII result in loss of binding activity. Analysis of the mutagenesis results together with multiple sequence alignments of a family of proteins that interacts with IIAGlc provides the basis for developing two regions of consensus sequence in those partner proteins necessary for binding to IIAGlc. The requirement for two interaction regions is interpreted in the regulatory framework of a substrate-dependent conformational change that brings those two regions into an orientation optimal for binding IIAGlc.

Amino Acid Sequence↗

PubMed1999

Proximity between periplasmic loops in the lactose permease of Escherichia coli as determined by site-directed spin labeling.

Site-directed thiol cross-linking indicates that the first periplasmic loop (loop I/II) in the lactose permease of Escherichia coli is in close proximity to loops VII/VIII and XI/XII [Sun, J., and Kaback, H. R. (1997) Biochemistry 36, 11959-11965]. To determine whether thiol cross-linking reflects proximity as opposed to differences in the reactivity and/or dynamics of the Cys residues that undergo cross-linking, single-Cys mutants in loops I/II, VII/VIII, and XI/XII and double-Cys mutants in loop I/II and VII/VIII or XI/XII were purified and labeled with a sulfhydryl-specific nitroxide spin label. The labeled mutants were then analyzed by electron paramagnetic resonance (EPR) spectroscopy, and interspin distance was estimated from the extent of line shape broadening in the double-labeled proteins. Out of six paired double-Cys mutants that exhibit thiol cross-linking, five display significant spin-spin interaction. Furthermore, there is a qualitative correlation between distances estimated by site-directed cross-linking and EPR. Taken as a whole, the results are consistent with the conclusion that site-directed thiol cross-linking is primarily a reflection of proximity.

Cross-Linking Reagents↗

PubMed1999

Helix packing in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking: helix I is close to helices V and XI.

Coexpression of lacY gene fragments encoding the first two transmembrane domains and the remaining 10 transmembrane domains complement in the membrane and catalyze active lactose transport [Wrubel, W., Stochaj, U., et al. (1990) J. Bacteriol. 172, 5374-5381]. Accordingly, a plasmid encoding contiguous, nonoverlapping permease fragments with a discontinuity in the cytoplasmic loop between helices II and III (loop II/III) was constructed (N2C10 permease). When Phe27 (helix I) is replaced with Cys, cross-linking is observed with two native Cys residues, Cys148 (helix V) and Cys355 (helix XI). Cross-linking of a Cys residue at position 27 to Cys148 occurs with N,N'-o-phenylenedimaleimide (o-PDM; rigid 6 A), with N,N'-p-phenylenedimaleimide (p-PDM; rigid 10 A), or with 1,6-bis(maleimido)hexane (BMH; flexible 16 A). On the other hand, with the Phe27-->Cys/Cys355 pair, cross-linking is observed with p-PDM or BMH but not o-PDM. In neither case is cross-linking observed with iodine. It is suggested that a Cys residue at position 27 is within 6-10 A from Cys148 and about 10 A from Cys355. The results provide evidence for proximity between helix I and helices V or XI in the tertiary structure of the permease. In addition, the findings are consistent with other results [Venkatesan, P., Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] indicating that Glu126 (helix IV) and Arg144 (helix V) are within the membrane, rather than at the membrane-water interface on the cytoplasmic face.

Biological Transport, Active↗

PubMed1999

Tertiary contacts of helix V in the lactose permease determined by site-directed chemical cross-linking in situ.

The six N-terminal transmembrane helices (N6) and the six C-terminal transmembrane helices (C6) in lactose permease, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices V and VII, VIII, or X was studied by thiol-specific chemical cross-linking. The results demonstrate that Cys residues in the periplasmic half of helix V cross-link with Cys residues in the periplasmic half of helix VII. In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices V and VII. Moreover, Cys residues on one entire face of helix V cross-link with Cys residues on one face of helix VIII. Finally, paired Cys residues at the cytoplasmic ends of helices V and X cross-link, but no cross-linking is observed when paired Cys residues are placed at the periplasmic ends of the two helices. Taken together, the results indicate that the periplasmic halves of helices V and VII are in close proximity and that the two helices tilt away from one another toward the cytoplasmic side of the membrane. Furthermore, helices V and VIII are in close proximity throughout their lengths and do not tilt appreciably with respect to one another, and helices V and X are in close proximity at the cytoplasmic but not at the periplasmic face of the membrane.

Amino Acid Substitution↗

PubMed1999

Site-directed chemical cross-linking demonstrates that helix IV is close to helices VII and XI in the lactose permease.

The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease, each containing a single-Cys residue, were coexpressed, and proximity was studied. Paired Cys residues in helices IV (positions 114, 116, 119, 122, 125, or 129) and VII (227, 231, 232, 234, 235, 238, 239, 242, 243, 245, or 246) or XI (350, 353, 354, 357, 361, or 364) were tested for cross-linking in the presence of two rigid homobifunctional thiol-specific cross-linkers, N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N,N'-p-phenylenedimaleimide (p-PDM; 10 A). Cys residues in the middle of helix IV (position 119 or 122) cross-link to Cys residues in the middle of helix VII (position 238, 239, 242, or 243). In contrast, no cross-linking is evident with paired Cys residues at either end of helix IV (position 114, 116, 125, or 129) or helix VII (position 227, 231, 232, 234, 235, 245, or 246). On the other hand, Cys residues in the cytoplasmic half of helix IV (position 125 or 129) cross-link with Cys residues in the cytoplasmic half of helix XI (position 350, 353, or 354), while paired Cys residues at the periplasmic ends of the two helices do not cross-link. The results indicate that helices IV and VII cross in a scissors-like manner with the cytoplasmic end of helix IV tilting toward helix XI.

Amino Acid Sequence↗

PubMed1999

Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli.

Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx. In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes. Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax. Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substrate protection against [14C]-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca. 80 microM). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity.

Amino Acid Substitution↗

PubMed1999

Two-dimensional crystallization of Escherichia coli lactose permease.

A chimeric protein consisting of lactose permease with cytochrome b562 in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L6cytb562/417H6 or "red permease") was overexpressed in Escherichia coli and isolated by nickel affinity chromatography after solubilization with dodecyl-beta,d-maltopyranoside. Red permease was then reconstituted in the presence of phospholipids, yielding densely packed vesicles and well-ordered two-dimensional (2D) crystals as shown by electron microscopy of negatively stained specimens. Single-particle analysis of 16 383 protein particles in densely packed vesicles reveals a 5.4-nm-long trapeziform protein of 4.1 to 5.1 nm width, with a central stain-filled indentation. Depending on reconstitution conditions, trigonal and rectangular crystallographic packing arrangements of these elongated particles assembled into trimers are observed. The best ordered 2D crystals exhibit a rectangular unit cell, of dimensions a = 9.9 nm, b = 17.4 nm, that houses two trimeric complexes. Projection maps calculated to a resolution of 2 nm show that these crystals consist of two layers.

Carrier Proteins↗