PubMed Health⌕ Search

Biomedical subjects

H R Kaback

Publications and source records attributed to H R Kaback.

At least 19 recordsLinked to original sources
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↗
PubMed1995

Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli.

Site-directed excimer fluorescence indicates that Glu269 (helix VIII) and His322 (helix X) in the lactose permease of Escherichia coli lie in close proximity [Jung, K., Jung, H., Wu, J., Privé, G.G., & Kaback, H.R. (1993) Biochemistry 32, 12273]. In this study, Glu269 was replaced with His in wild-type permease, leading to the presence of bis-His residues between helices VIII and X. Wild-type and Glu269-->His permease containing a biotin acceptor domain were purified by monomeric avidin affinity chromatography, and binding of Mn2+ was studied by electron paramagnetic resonance (EPR) spectroscopy. The amplitude of the Mn2+ EPR spectrum is reduced by the Glu269-->His mutant, while no change is observed in the presence of wild-type permease. The Glu269-->His mutant contains a single binding site for Mn2+ with a KD of about 43 microM, and Mn2+ binding is pH dependent with no binding at pH 5.0, stoichiometric binding at pH 7.5, and a midpoint at about pH 6.3. The results confirm the conclusion that helices VIII and X are closely opposed in the tertiary structure of lac permease and provide a novel approach for studying helix proximity, as well as solvent accessibility, in polytopic membrane proteins.

Binding Sites↗
PubMed1995

Active transport by the CscB permease in Escherichia coli K-12.

The cscB gene encoding a putative sucrose permease from Escherichia coli EC3132 [Bockmann, J., Heuel, H. & Lengeler, J.W. (1992) Mol. Gen. Genet. 235, 22-32] was placed under control of the lacZ promoter/operator and overexpressed. Cells of E. coli K-12 expressing cscB gene product from a high copy number plasmid accumulate sucrose against a concentration gradient with an apparent Km of about 1 mM and a Vmax of approximately 23 nmol/min/mg protein. In contrast, transport of lactose or melibiose is not observed. Epitope-tagging and immunological analysis reveal that concomitant with increased transport activity, a protein with an apparent M(r) of 33-34 kDa appears in the cytoplasmic membrane. The results provide direct support for the contention that the cscB gene encodes a sucrose permease and provide the basis for further molecular analysis.

Base Sequence↗
PubMed1994

Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites.

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331-->Cys permease was then purified and studied in dodecyl-beta,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331-->Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (< 0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd approximately equal to 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331-->Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 -->Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10 mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The role of transmembrane domain III in the lactose permease of Escherichia coli.

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.

Amino Acid Sequence↗
PubMed1992

Insertional mutagenesis of hydrophilic domains in the lactose permease of Escherichia coli.

The lactose permease of Escherichia coli is a membrane transport protein postulated to contain a hydrophilic N terminus (hydrophilic domain 1), 12 hydrophobic transmembrane alpha-helices that traverse the membrane in zigzag fashion connected by hydrophilic domains, and a hydrophilic C terminus (hydrophilic domain 13). To test whether the hydrophilic domains are important for function, each domain was independently disrupted by insertion of two or six contiguous histidine residues, and the mutants were characterized with respect to initial rate of lactose transport and steady-state level of accumulation. Remarkably, histidine insertions into 10 out of 13 hydrophilic domains result in molecules that catalyze lactose accumulation effectively, although the initial rate of transport is compromised in certain cases. In contrast, insertions into hydrophilic domain 3, 9, or 10 cause a marked decrease in transport activity. As judged by immunoblots and [35S]methionine pulse-chase experiments, diminished activity is not due to decreased expression of the mutated permeases, defective insertion into the membrane, or increased rates of proteolysis after insertion. The results (i) suggest that most of the hydrophilic domains in the permease do not play an essential role in the transport mechanism and (ii) focus on the region of the permease containing putative helices IX and X as being particularly important for activity.

Amino Acid Sequence↗
PubMed1992

Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coli.

Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual replacements for Asp-237, Asp-240, Glu-269, Arg-302, Lys-319, His-322, Glu-325, or Lys-358 abolish active lactose transport. When Asp-237 and Lys-358 are simultaneously replaced with Cys and/or Ala, however, high activity is observed. Therefore, when either Asp-237 or Lys-358 is replaced with a neutral residue, leaving an unpaired charge, the permease is inactivated, but neutral replacement of both residues yields active permease [King, S. C., Hansen, C. L. & Wilson, T. H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Remarkably, moreover, when Asp-237 is interchanged with Lys-358, high activity is observed. The observations provide a strong indication that Asp-237 and Lys-358 interact to form a salt bridge. In addition, the data demonstrate that neither residue nor the salt bridge plays an important role in the transport mechanism. Thirteen additional double mutants were constructed in which a negative and a positively charged residue were replaced with Cys. Only Asp-240-->Cys/Lys-319-->Cys exhibits significant activity, accumulating lactose to 25-30% of the steady state observed with C-less permease. Replacing either Asp-240 or Lys-319 individually with Ala also inactivates the permease, but double mutants with neutral substitutions (Cys and/or Ala) at both positions exhibit essentially the same activity as Asp-240-->Cys/Lys-319-->Cys. In marked contrast to Asp-237 and Lys-358, interchanging Asp-240 and Lys-319 abolishes active lactose transport. The results demonstrate that Asp-240 and Lys-319, like Asp-237 and Lys-358, interact functionally and may form a salt bridge. However, the interaction between Asp-240 and Lys-319 is clearly more complex than the interaction between Asp-237 and Lys-358. In any event, the findings suggest that putative transmembrane helix VII lies next to helices X and XI in the tertiary structure of lactose permease.

Amino Acid Sequence↗
PubMed1992

Site-directed mutagenesis of lysine 319 in the lactose permease of Escherichia coli.

Lys319, which is on the same face of putative helix X as His322 and Glu325 in the lactose permease of Escherichia coli, has been replaced with Leu by oligonucleotide-directed, site-specific mutagenesis. Although previous experiments suggested that the mutation does not alter permease activity, we report here that K319L permease is unable to catalyze active lactose accumulation or lactose efflux down a concentration gradient. The mutant does catalyze facilitated influx down a concentration gradient at a significant rate; however, the reaction occurs without concomitant H+ translocation. The mutant also catalyzes equilibrium exchange at about 50% of the wild-type rate, but it exhibits poor counterflow activity. Finally, flow dialysis and photoaffinity labeling experiments with p-nitrophenyl alpha-D-galactopyranoside indicate that K319L permease probably has a markedly decreased affinity for substrate. The alterations described are not due to diminished levels of the mutated protein in the membrane, since immunological studies reveal comparable amounts of permease in wild-type and K319L membranes. It is proposed that Lys319, like Arg302, His322, and Glu325, plays an important role in active lactose transport, as well as substrate recognition.

Affinity Labels↗
PubMed1992

Melibiose permease of Escherichia coli: mutation of histidine-94 alters expression and stability rather than catalytic activity.

Previous studies utilizing site-directed mutagenesis [Pourcher et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 468-472] indicate that out of seven histidinyl residues in the melibiose (mel) permease of Escherichia coli, only His94 is important. The role of His94 has now been investigated by replacing the residue with Asn, Gln, or Arg. Cells expressing mel permease with Asn94 or Gln94 retain 30% or 20% of wild-type activity, respectively, and surprisingly, immunological assays demonstrate that diminished transport activity is due to a proportional reduction in the amount of permease in the membrane. Moreover, kinetic analyses of transport and ligand binding studies with right-side-out membrane vesicles indicate that both substrate recognition and turnover (kcat) are comparable in the mutant permeases and the wild-type. Mel permease with Arg in place of His94 also binds ligand and catalyzes sugar accumulation, but only when the cells are grown at 30 degrees C, and evidence is presented that Arg94 permease is inactivated at 37 degrees C. Finally, labeling studies demonstrate that expression and/or insertion of the permease, but not degradation, is strongly dependent on the amino acid present at position 94 and temperature. The findings indicate that an imidazole group at position 94 is required for proper insertion and stability of mel permease, but not for transport activity per se. Since replacement of the other six histidinyl residues in mel permease with Arg has little or no effect on transport activity, it is concluded that histidinyl residues do not play a direct role in the mechanism of this secondary transport protein.

Base Sequence↗
PubMed1992

The N-terminal 22 amino acid residues in the lactose permease of Escherichia coli are not obligatory for membrane insertion or transport activity.

When the lactose (lac) permease of Escherichia coli is expressed from the lac promoter at relatively low rates, deletion of amino acid residues 2-8 (delta 7) or 2-9 (delta 8) from the hydrophilic N terminus has a relatively minor effect on the ability of the permease to catalyze active lactose transport. Activity is essentially abolished, however, and the permease is hardly detected in the membrane when two additional amino acid residues are deleted (delta 10), and mutants deleted of residues 2-23 (delta 22) or 2-39 (delta 38) also exhibit no activity and are not inserted into the membrane. Dramatically, when the defective deletion mutants are overexpressed at high rates via the T7 promoter, delta 10 and delta 22 are inserted into the membrane in a stable form and catalyze active lactose transport in a highly significant manner, whereas delta 38 is hardly detected in the membrane and exhibits no activity. Interestingly, a fusion protein consisting of delta 38 and the ompA leader peptide is inserted into the membrane but exhibits no transport activity. The results indicate that the N-terminal hydrophilic domain of lac permease and the N-terminal half of the first putative transmembrane alpha-helix are not mandatory for either membrane insertion or transport activity.

Amino Acid Sequence↗
PubMed1992

Evidence that the final turn of the last transmembrane helix in the lactose permease is required for folding.

Although truncation of the hydrophilic C-terminal tail of the lactose (lac) permease of Escherichia coli (residues 401-417) has no significant effect on membrane insertion, stability, or transport activity, sequential substitution of stop codons for amino acid codons 398-401 leads to a progressive increase in transport activity and in the lifetime of the permease in the membrane (McKenna, E., Hardy, D., Pastore, J. C., and Kaback, H. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969-2973). Thus, either the last turn of putative helix XII or the region immediately distal to helix XII is important for proper folding, and hence, activity and resistance to proteolysis. In an effort to determine whether this 3-4-amino acid sequence comprises the final turn of the last transmembrane helix of the permease or the beginning of the hydrophilic C-terminal tail, we deleted residues 401-417 and replaced amino acid residues 397-400 with either 4 Leu residues ("helix making") or Gly-Pro-Gly-Pro ("helix breaking"). Permease with 4 Leu residues at positions 397-400 is fully functional with respect to transport and completely stable, as judged by [35S]methionine labeling experiments. In marked contrast, permease with Gly-Pro-Gly-Pro at the same positions exhibits minimal activity and is unstable. The results imply that the amino acid sequence ... Val397Phe398Thr399 Leu400 ... in lac permease may comprise the last turn of transmembrane helix XII, rather than the beginning of the C-terminal tail.

Amino Acid Sequence↗