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H R Kaback

Publications and source records attributed to H R Kaback.

At least 55 records · Page 3Linked to original sources
PubMed1998

Tilting of helix I and ligand-induced changes in the lactose permease determined by site-directed chemical cross-linking in situ.

The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of lactose permease, each with a single Cys residue, were co-expressed, and cross-linking was studied. The proximity of paired Cys residues in helices I (positions 11, 14, 15, 18, 25, 28, 29, or 32) and VII (positions 227, 231, 232, 234, 235, 238, 239, 241, 242, 245, or 246) was studied by using homobifunctional thiol-specific chemical linkers of different lengths and chemical properties. The results demonstrate that Cys residues on one face of the periplasmic half of helix I (positions 32, 29, 28, or 25) cross-link to Cys residues on one face of the periplasmic half of helix VII (242 or 245). In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices I (positions 11, 14, 15, or 18) and VII (positions 227, 230, 231, 232, 234, 235, 238, or 239). The results indicate that helices I and VII are in close proximity only at their periplasmic halves. Ligand binding decreases cross-linking efficiency of the Cys pair 28/245 or 25/242 with N, N'-o-phenylenedimaleimide (rigid 6 A) and increases efficiency with N,N'-p-phenylenedimaleimide (rigid 10 A) or 1,6-bismaleimidohexane (flexible 16 A), indicating that the inter-thiol distance is about 6 A in the absence of ligand and that ligand binding increases the distance up to 10 A. The inter-thiol distance for Cys pairs 29/245 or 32/245 is less than 6 A in the absence of ligand, and in the presence of ligand, distance increases to between 6 and 10 A. Taken together, the results indicate that ligand binding induces a translational or scissors-like rigid body movement of helix I and/or VII at the periplasmic interface between the helices.

Cross-Linking Reagents↗
PubMed1998

In vitro biotinylation provides quantitative recovery of highly purified active lactose permease in a single step.

Consler et al. [Consler, T. G., Persson, B. L., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6934-6938] described a one-step purification of lactose permease, a hydrophobic membrane transport protein, from Escherichia coli. Permease constructs containing a biotin acceptor domain are biotinylated in vivo, followed by solubilization and avidin affinity purification. Although a high degree of purity is obtained, only about 15-20% of the permease is recovered due to incomplete biotinylation. In this communication, a simple modification is described that allows quantitative recovery of highly purified permease. Membranes containing permease with the biotin acceptor domain from the Klebsiella pneumoniae oxaloacetate decarboxylase are extracted with 5 M urea or treated with dicyclohexylcarbodiimide to inactivate F1/Fo ATPase and biotinylated in vitro with biotin ligase, ATP and d-biotin. Subsequently, the membranes are harvested, washed to remove free biotin and solubilized with 2% n-dodecyl-beta-D-maltopyranoside. Biotinylated permease is then purified in one step by affinity chromatography on monomeric avidin-Sepharose. The purified material is homogeneous and exhibits full activity with respect to ligand binding and counterflow.

Anilino Naphthalenesulfonates↗
PubMed1998

Transmembrane helix tilting and ligand-induced conformational changes in the lactose permease determined by site-directed chemical crosslinking in situ.

The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease of Escherichia coli, each with a Cys residue, were co-expressed independently, and crosslinking was studied. Proximity of paired Cys residues in helices II (position 49, 52, 53, 56, 57, 60, 63 or 67) and VII (position 227, 230, 231, 234, 238, 241, 242 or 245) or XI (position 350, 353, 354, 357, 361 or 364) was examined by using two homobifunctional thiol-specific crosslinking agents of different lengths (6 or 10 A). The results demonstrate that a Cys residue placed in the periplasmic half of helix II (position 49, 52, 53 or 57) crosslinks to Cys residues in the periplasmic half of helix VII (position 241, 242 or 245). In contrast, no crosslinking is evident with paired-Cys residues in the cytoplasmic halves of helices II (position 60, 63 or 67) and VII (position 227, 230, 231, 234 or 238). Remarkably, a Cys residue in the cytoplasmic half of helix II (position 60, 63 or 67) crosslinks with a Cys residue in the cytoplasmic half of helix XI (position 350, 353 or 354), while paired-Cys residues at positions in the periplasmic halves of the two helices do not crosslink. Therefore, helix II is tilted in such a manner that the periplasmic end is close to helix VII, and the cytoplasmic end is close to helix XI. Furthermore, ligand-binding alters the crosslinking efficiency of paired-Cys residues in helices II and VII or XI, indicating that both interfaces are conformationally active. The results are consistent with the conclusion that ligand-binding induces a scissors-like movement of helices II and VII that increases interhelical distance by 3 to 4 A at the periplasmic ends and decreases the distance by 3 to 4 A at the approximate middle of the two transmembrane helices.

Cross-Linking Reagents↗
PubMed1998

The substrate-binding site in the lactose permease of Escherichia coli.

Site-directed N-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys or His does not alter reactivity, indicating that Glu-126 and Arg-144 are charge-paired. Importantly, although alkylation of Cys-148 is blocked by ligand in wild-type permease, no protection whatsoever is observed with any of the Glu-126 or Arg-144 mutants. Site-directed fluorescence with 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS) in mutant Val-331 --> Cys was also studied. In marked contrast to Val-331 --> Cys permease, ligand does not alter MIANS reactivity in mutant Glu-126 --> Ala/Val-331 --> Cys, Arg-144 --> Ala/Val-331 --> Cys, or Arg-144 --> Lys/Val-331 --> Cys and does not cause either quenching or a shift in the emission maximum of the MIANS-labeled mutants. However, mutation Glu-126 --> Ala or Arg-144 --> Ala and, to a lesser extent, Arg-144 --> Lys cause a red-shift in the emission spectrum and render the fluorophore more accessible to I-. The results demonstrate that Glu-126 and Arg-144 are irreplaceable for substrate binding and suggest a model for the substrate-binding site in the permease. In addition, the findings are consistent with the notion that alterations in the substrate translocation pathway at the interface between helices IV and V are transmitted conformationally to the H+ translocation pathway at the interface between helices IX and X.

Anilino Naphthalenesulfonates↗
PubMed1998

Ligand-induced changes in periplasmic loops in the lactose permease of Escherichia coli.

N- and C-terminal halves of lactose permease, each with a single-Cys residue in a periplasmic loop, were coexpressed, and cross-linking was studied in the presence or absence of ligand. A Cys residue at position 36 between transmembrane helices I and II (loop I/II) forms a disulfide spontaneously with a Cys residue at position 253, 254, 255, or 256 in loop VII/VIII. Moreover, in the presence of o-phenanthroline-copper, a Cys residue at position 42 in loop I/II forms a disulfide with a Cys residue at position 253, 254, 257, or 258 in loop VII/VIII. Changes in the rate of cross-linking are also observed in the presence of substrate, suggesting that ligand binding induces movement between loops I/II and VII/VIII such that positions 253-256 are brought closer to position 36 or 42, while positions 257 and 258 move away from position 42.

Amino Acid Substitution↗
PubMed1998

Sulfhydryl oxidation of mutants with cysteine in place of acidic residues in the lactose permease.

To examine further the role of charge-pair interactions in the structure and function of lactose permease, Asp237 (helix VII), Asp240 (helix VII), Glu126 (cytoplasmic loop IV/V), Glu269 (helix VIII), and Glu325 (helix X) were replaced individually with Cys in a functional mutant devoid of Cys residues. Each mutant was then oxidized with H2O2 in order to generate a sulfinic and/or sulfonic acid at these positions. Due to the isosteric relationship between aspartate and sulfinate, in particular, and the lower pKa of the sulfinic and sulfonic acid side chains, oxidized derivatives of Cys are useful probes for examining the role of carboxylates. Asp237-->Cys or Asp240-->Cys permease is inactive, as shown previously, but H2O2 oxidation restores activity to an extent similar to that observed when a negative charge is reintroduced by other means. Glu126-->Cys, Glu269-->Cys, or Glu325-->Cys permease is inactive, but oxidation does not restore active lactose transport. The data are consistent with previous observations indicating that Asp237 and Asp240 are not critical for active lactose transport, while Glu126, Glu269, and Glu325 are irreplaceable. Although Glu269-->Cys permease does not transport lactose, the oxidized mutant exhibits significant transport of beta,D-galactosylpyranosyl 1-thio-beta,D-galactopyranoside, a property observed with Glu269-->Asp permease. The observation supports the idea that an acidic residue at position 269 is important for substrate recognition. Finally, oxidized Glu325-->Cys permease catalyzes equilibrium exchange with an apparent pKa of about 6.5, more than a pH unit lower than that observed with Glu325-->Asp permease, thereby providing strong confirmatory evidence that a negative charge at position 325 determines the rate of translocation of the ternary complex between the permease, substrate, and H+.

Amino Acid Substitution↗
PubMed1998

Fourier transform infrared spectroscopy reveals a rigid alpha-helical assembly for the tetrameric Streptomyces lividans K+ channel.

The structure of the tetrameric K+ channel from Streptomyces lividans in a lipid bilayer environment was studied by polarized attenuated total reflection Fourier transform infrared spectroscopy. The channel displays approximately 43% alpha-helical and 25% beta-sheet content. In addition, H/D exchange experiments show that only 43% of the backbone amide protons are exchangeable with solvent. On average, the alpha-helices are tilted 33 degrees normal to the membrane surface. The results are discussed in relationship to the lactose permease of Escherichia coli, a membrane transport protein.

Lipid Bilayers↗
PubMed1998

Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli.

Three double-Cys mutant pairs--Ala273-->Cys/Met299-->Cys, Thr266-->Cys/Ile303-->Cys, and Thr266-->Cys/Ser306-->Cys--were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273-->Cys/Met299-->Cys, but not with the single-Cys mutants nor with mutants Thr266-->Cys/Ile303-->Cys or Thr266-->Cys/Ser306-->Cys. Furthermore, spin-spin interaction is also observed with mutant Ala273-->Cys/Met299-->Cys, but only after the permease is reconstituted into proteoliposomes. The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.

Alanine↗
PubMed1998

Helix packing in the lactose permease determined by metal-nitroxide interaction.

The magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances within proteins [Voss, J., Salwinski, L., Kaback, H. R., and Hubbell, W. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12295-12299; Voss, J., Hubbell, W. L., and Kaback, H. R. (1995) Proc. Natl. Acad. Sci. U.S. A. 92, 12300-12303], and the approach is utilized here to measure helix proximities in the lactose permease of Escherichia coli. A high-affinity divalent metal binding site was created by replacing Arg302 (helix IX) and Glu325 (helix X) with His residues in permease mutants containing single Cys residues in helices II, V, or VII and a biotin acceptor domain to facilitate purification. Mutant proteins were purified by avidin affinity chromatography, labeled specifically with a nitroxide free radical and investigated by electron paramagnetic resonance spectroscopy in the absence or presence of Cu(II). Spectral broadening due to bound Cu(II) was used to estimate distances between the metal center and the spin-labeled side chains. For each of the transmembrane domains probed, the variation in interspin distance with sequence position is consistent with an alpha-helical structure. The measured distances were also used to construct a model that is in good agreement with packing data obtained from other approaches.

Bacterial Proteins↗
PubMed1998

Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins.

The entire lactose permease of Escherichia coli, a polytopic membrane transport protein that catalyzes beta-galactoside/H+ symport, has been subjected to Cys-scanning mutagenesis in order to determine which residues play an obligatory role in the mechanism and to create a library of mutants with a single-Cys residue at each position of the molecule for structure/function studies. Analysis of the mutants has led to the following: 1) only six amino acid side chains play an irreplaceable role in the transport mechanism; 2) positions where the reactivity of the Cys replacement is increased upon ligand binding are identified; 3) positions where the reactivity of the Cys replacement is decreased by ligand binding are identified; 4) helix packing, helix tilt, and ligand-induced conformational changes are determined by using the library of mutants in conjunction with a battery of site-directed techniques; 5) the permease is a highly flexible molecule; and 6) a working model that explains coupling between beta-galactoside and H+ translocation. structure-function relationships in polytopic membrane proteins.

Alkylation↗
PubMed1997

Site-directed spin-labeling of transmembrane domain VII and the 4B1 antibody epitope in the lactose permease of Escherichia coli.

Functional lactose permease mutants containing single Cys residues at positions 233-255 and a biotin acceptor domain at the C terminus were solubilized in dodecyl beta-d-maltopyranoside and purified by avidin affinity chromatography. Each mutant protein was derivatized with a thiol-selective nitroxide reagent and examined by conventional and power saturation electron paramagnetic resonance spectroscopy (EPR). The EPR spectral line shapes and the influence of nonpolar O2 or polar potassium chromium oxalate relaxation agents on the saturation behavior of the spin-labeled proteins were measured in order to obtain information on the mobility of the spin-labeled side chains and their accessibility to the relaxation agents, respectively. The results provide evidence that residues Ser233-Asn246 are within the hydrophobic core of the membrane and that Phe247 is at the lipid headgroup-solvent interface. Along with Phe247, Phe250 and Gly254 are also surface-exposed, as indicated by studies on the epitope for monoclonal antibody 4B1 [Sun, J., Wu, J., Carasco, N., and Kaback, H. R. (1996) Biochemistry 35, 990-998]. Furthermore, the nitroxide-labeled intramembrane Cys replacements exhibit variations in mobility and accessibility that are consistent with the conclusion that TM VII is an alpha-helix in contact with surrounding helices in the tertiary structure of the permease.

Amino Acid Sequence↗
PubMed1997

Topology of allosteric regulation of lactose permease.

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.

Allosteric Regulation↗
PubMed1997

Cysteine-scanning mutagenesis of helix IV and the adjoining loops in the lactose permease of Escherichia coli: Glu126 and Arg144 are essential. off.

Cys-scanning mutagenesis has been applied to the remaining 45 residues in lactose permease that have not been mutagenized previously (from Gln100 to Arg144 which comprise helix IV and adjoining loops). Of the 45 single-Cys mutants, 26 accumulate lactose to > 75% of the steady state observed with Cys-less permease, and 14 mutants exhibit lower but significant levels of accumulation (35-65% of Cys-less permease). Permease with Phe140-->Cys or Lys131-->Cys exhibits low activity (15-20% of Cys-less permease), while mutants Gly115-->Cys, Glu126-->Cys and Arg144-->Cys are completely unable to accumulate the dissacharide. However, Cys-less permease with Ala or Pro in place of Gly115 is highly active, and replacement of Lys131 or Phe140 with Cys in wild-type permease has a less deleterious effect on activity. In contrast, mutant Glu126-->Cys or Arg144-->Cys is inactive with respect to both uphill and downhill transport in either Cys-less or wild-type permease. Furthermore, mutants Glu126-->Ala or Gln and Arg144-->Ala or Gln are also inactive in both backgrounds, and activity is not rescued by double neutral replacements or inversion of the charged residues at these positions. Finally, a mutant with Lys in place of Arg144 accumulates lactose to about 25% of the steady state of wild-type, but at a slow rate. Replacement of Glu126 with Asp, in contrast, has relatively little effect on activity. None of the effects can be attributed to decreased expression of the mutants, as judged by immunoblot analysis. Although the activity of most of the single-Cys mutants is unaffected by N-ethylmaleimide, Cys replacement at three positions (Ala127, Val132, or Phe138) renders the permease highly sensitive to alkylation. The results indicate that the cytoplasmic loop between helices IV and V, where insertional mutagenesis has little effect on activity [McKenna, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11954-11958], contains residues that play an important role in permease activity and that a carboxyl group at position 126 and a positive charge at position 144 are absolutely required.

Amino Acid Sequence↗
PubMed1997

Alanine insertion scanning mutagenesis of lactose permease transmembrane helices.

A priori, single residue insertions into transmembrane helices are expected to be highly disruptive to protein structure and function. We have carried out a systematic analysis of the phenotypes associated with Ala insertions into transmembrane helices in lactose permease, a multispanning Escherichia coli inner membrane protein. Insertion of alanine into the center of 7 transmembrane helices was found to abolish stable integration of lactose permease into the membrane or uphill lactose transport. A more detailed Ala insertion scan was made of transmembrane helix III. The results pin-point a central region of approximately 2 helical turns that is crucial for lactose permease stability and/or activity. A Trp scan in this region identified 2 residues essential for lactose permease stability. From these results, it appears that transmembrane helices have differential sensitivities to single residue insertions and that such mutations may be useful for identifying structurally and/or functionally important helix segments.

Alanine↗
PubMed1997

Ligand-induced movement of helix X in the lactose permease from Escherichia coli: a fluorescence quenching study.

Five single-Trp mutants were constructed by replacing Val315, Leu318, Val326, Leu329, or Val331 with Trp in transmembrane helix X of a functional lactose permease mutant devoid of Trp residues (Trp-less permease). Taking into account expression levels, each single-Trp permease except for Val331-->Trp exhibits significant activity. The intrinsic fluorescence emission of each single-Trp mutant does not change significantly after addition of beta-d-galactopyranosyl 1-thio-beta-d-galactopyranoside (TDG), indicating that ligand induces little change in the microenvironment of the Trp residues. However, fluorescence quenching studies with the brominated detergent 7,8-dibromododecyl beta,d-maltoside (BrDM) demonstrate that a Trp residue in place of Val315, Val326, or Val331 becomes less accessible to BrDM in the presence of TDG, while a Trp residue in place of Leu318 or Leu329 becomes more accessible. Acrylamide quenching studies with Leu318-->Trp and Val331-->Trp permeases or 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS)-labeled Thr320-->Cys and Glu325-->Cys permeases indicate that positions 318 and 325 also become more accessible to a hydrophobic environment in the presence of TDG, while positions 320 and 331 become less accessible. The findings are consistent with a recently proposed mechanism for energy coupling in lactose permease [Kaback, H. R. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 5539-5543] in which substrate binding causes a conformational change resulting in movement of Glu325 to a nonpolar environment with a dramatic increase in pKa.

Amino Acid Sequence↗
PubMed1997

Arginine 302 (helix IX) in the lactose permease of Escherichia coli is in close proximity to glutamate 269 (helix VIII) as well as glutamate 325.

By using a variety of biochemical and biophysical approaches, a helix packing model for the lactose permease of Escherichia coli has been proposed in which the four residues that are irreplaceable with respect to coupling are paired--Glu269 (helix VIII) with His322 (helix X) and Arg302 (helix XI) with Glu325 (helix X). In addition, the substrate translocation pathway is located at the interface between helices V and VIII, which is in close vicinity to the four essential residues. Based on this structural information and functional studies of mutants in the four irreplaceable residues, a molecular mechanism for energy coupling in the permease has been proposed [Kaback, H. R. (1997) Pro.c Natl. Acad. Sci. U.S.A. 94, 5539]. The principle idea of this model is that Arg302 interacts with either Glu325 or Glu269 during turnover. Evidence that Arg302 is in close proximity with Glu325 has been presented [Jung, K., Jung, H., Wu, J., Prive, G. G., & Kaback, H. R. (1993) Biochemistry 32, 12273; He, M. M., Voss, J., Hubbell, W. L., & Kaback, H. R. (1995) Biochemistry 34, 15667]; however, the proximity of Arg302 to Glu269 has not been examined. In this report, it is shown by two methods that Arg302 is also close to Glu269: (i) permease with Glu269-->His, Arg302-->His, and His322-->Phe binds Mn2+ with high affinity at pH 7.5, but not at pH 5.5; and (ii) site-directed spin-labeling of the double Cys mutant Glu269-->Cys/Arg302-->Cys exhibits spin-spin interaction with an interspin distance of about 14-16 A. In addition, the spin-spin interaction is stronger and interspin distance shorter after the permease is reconstituted into proteoliposomes. Taken as a whole, the data are consistent with the idea that Arg302 may interact with either Glu325 or Glu269 during turnover.

Amino Acid Sequence↗
PubMed1997

Interaction between residues Glu269 (helix VIII) and His322 (helix X) of the lactose permease of Escherichia coli is essential for substrate binding.

Site-directed and Cys-scanning mutagenesis of the lactose permease of Escherichia coli reveals that as few as four residues--Glu269 (helix VIII), Arg302 (helix IV), His322 (helix X), and Glu325 (helix X)--are irreplaceable for coupling substrate and H+ translocation. Interestingly, the four residues are in close physical proximity, Glu269 interacting with His322 and Arg302 with Glu325. In addition, the substrate translocation pathway is located close to the four residues at the interface between helices V and VIII. To investigate the importance of the four residues and their interactions for substrate binding, mutation Glu269-->Asp, Glu269-->Gln, Arg302-->Ala, Arg302-->Lys, His322-->Ala, His322-->Phe, Glu325-->Asp, or Glu325-->Gln was introduced into single-Cys148 permease, where the reactivity of Cys with 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) is blocked by binding of substrate. The double mutants were purified, and the rates of MIANS labeling were measured in the absence or presence of beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG), lactose, or galactose at various concentrations. Remarkably, substrate binding by the Glu269 or His322 mutants is abolished or decreased dramatically, while binding by the Arg302 or Glu325 mutants is not altered. The observations are consistent with the notion that the interaction between Glu269 and His322 stabilizes the interface between helices V and VIII and thereby leads to binding of substrate.

Anilino Naphthalenesulfonates↗