Cys‐scanning mutagenesis: a novel approach to structure—function relationships in polytopic membrane proteins
The FASEB JournalPublished 1 October 1998
Stathis Frillingos, Miklós Sahin‐Tóth, Jianhua Wu, H. Ronald Kaback
Citations357
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SJR score1.32
SNIP0.89
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TL;DR
The entire lactose permease of Escherichia coli, a polytopic membrane transport protein that catalyzes β‐galactoside/H+ symport, has been subjected to Cys‐scanning mutagenesis 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.
Abstract
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.
Keywords
Biochemistry, Genetics and Molecular Biology
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Proceedings of the National Academy of SciencesInsertional mutagenesis of hydrophilic domains in the lactose permease of Escherichia coli.
38 Citations1992Eva J. McKenna, Dorothy Hardy +1 more
It is suggested that most of the hydrophilic domains in the permease do not play an essential role in the transport mechanism and focus on the region of the permeases containing putative helices IX and X as being particularly important for activity.
BiochemistryUse 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)
37 Citations1995Molly M. He, John C. Voss +2 more
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, which provides strong support for the helix packing model proposed.
Journal of Biological ChemistryCharacterization of the double mutant, Val-177/Asn-322, of the lactose permease.
37 Citations1990Robert J. Brooker
Direct measurements of H+ transport showed that the Val-177/Asn-322 mutant's defect in active accumulation is not due to a defect in the ability to transport H+ with lactose or methyl-beta-D-thiogalactopyranoside, and the role His-322 plays in H+ Transport, active accumulation of sugars, and sugar recognition is discussed.
Journal of BacteriologySuppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes
37 Citations1997Amy E. Jessen-Marshall, N J Parker +1 more
The notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane is supported.
Protein ScienceThe role of helix VIII in the lactose permease of <i>Escherichia coli</i>: II. Site‐directed sulfhydryl modification
34 Citations1997Stathis Frillingos, H. Ronald Kaback
The findings indicate that a portion of one face of helix VIII, which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand‐induced conformational change.
BiochemistryDetermination of a Transmembrane Segment Using Cysteine-Scanning Mutants of Transposon Tn10-Encoded Metal−Tetracycline/H<sup>+</sup> Antiporter
34 Citations1996Tomomi Kimura, Masayo Suzuki +2 more
The results for 37 cysteine-scanning mutants around putative transmembrane helix 9 indicate that the residues from 266 to 275 and from 296 to 298 form water extruding loops, while residues from 276 to 295 and from 299 to 302 are embedded in the membrane.
Protein ScienceLigand‐Induced conformational changes in the lactose permease of <i>escherichia coli</i>: Evidence for two binding sites
33 Citations1994Jianhua Wu, Stathis Frillingos +2 more
Results provide evidence for the argument that lac permease has more than a single binding site and TDG binding to a higher affinity site quenches the fluorescence of MIANS‐labeled Val 331 → Cys permease, and occupation of a second lower affinity site causes position 331 to become more accessible from a hydrophobic environment.
Protein ScienceCysteine scanning mutagenesis of the N‐terminal 32 amino acid residues in the lactose permease of <i>Escherichia coli</i>
33 Citations1994Miklós Sahin‐Tóth, Bengt Persson +3 more
The results demonstrate that although no residue per se in this region is essential for activity, the structural integrity of the periplasmic half of the first transmembrane helix is important for active lactose transport.
Proceedings of the National Academy of SciencesSequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability.
33 Citations1991Eva J. McKenna, Dorothy Hardy +2 more
Stop codons have now been substituted sequentially for amino acid codons 396-401 in the lacY gene and the termination mutants were expressed from the plasmid pT7-5, indicating that either the last turn of putative helix XI or the region immediately distal to helix XII is important for proper folding and protection against proteolytic degradation.
Journal of Biological ChemistrySensitivity of efflux-driven carrier turnover to external pH in mutants of the Escherichia coli lactose carrier that have tyrosine or phenylalanine substituted for histidine-322. A comparison of lactose and melibiose.
33 Citations1990Steven C. King, T. Hastings Wilson
The modest effect of these mutations on the transport rate indicates that His-322 contributes a far smaller free energy increment to catalyzing of H+/galactoside cotransport than active site histidines contribute to catalyzed peptide bond hydrolysis in serine proteases.
BiochemistryProximity of Periplasmic Loops in the Lactose Permease of <i>Escherichia coli </i>Determined by Site-Directed Cross-Linking
33 Citations1997Jianzhong Sun, H. Ronald Kaback
Evidence is presented indicating that conformational flexibility between loops I/II and XI/XII may be important for permease turnover, and cross-linking of residues between loops is specific, indicating that these domains are not simply flexible, hydrophilic connections between helices that interact randomly.
BiochemistryUse 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 helixes X (Lys319) and XI (Lys358)
32 Citations1995Molly M. He, John C. Voss +2 more
The lactose permease of Escherichia coli contains two pairs of oppositely charged residues that interact functionally, Asp240 and Asp237, and single- and double-His replacement mutants at these positions have been constructed and characterized with respect to transport activity and Mn2+ binding.
Handbook of biological physicsChapter 10 The lactose permease of Escherichia coli: Past, present and future
32 Citations1996H. Ronald Kaback
Unequivocal support for the topological predictions of the 12-helix model is obtained by analyzing a large number of lac permease-alkaline phosphatase (lacY-phoA) fusions.
Journal of Biological ChemistrySite-specific alteration of cysteine 176 and cysteine 234 in the lactose carrier of Escherichia coli.
32 Citations1986Robert J. Brooker, T. Hastings Wilson
The mutant proteins are able to effectively transport galactosides, although the transport rates for lactose and methyl-beta-D-galactopyranoside are slightly reduced compared to the normal lactose carrier.
BiochemistryInteraction between Residues Glu269 (Helix VIII) and His322 (Helix X) of the Lactose Permease of <i>Escherichia coli</i> Is Essential for Substrate Binding
32 Citations1997Molly M. He, H. Ronald Kaback
Site-directed and Cys-scanning mutagenesis of the lactose permease of Escherichia coli reveals that as few as four residues are irreplaceable for coupling substrate and H+ translocation, 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.
BiochemistryMonoclonal Antibody 4B1 Alters the p<i>K</i><sub>a</sub> of a Carboxylic Acid at Position 325 (Helix X) of the Lactose Permease of <i>Escherichia coli</i>
30 Citations1996Stathis Frillingos, H. Ronald Kaback
P pH profiles for active transport, efflux, and equilibrium exchange are analyzed for wild-type permease and mutant Glu325-->Asp and 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.
BiochemistryCysteine-Scanning Mutagenesis of Transmembrane Domain XII and the Flanking Periplasmic Loop in the Lactose Permease of <i>Escherichia coli</i>
30 Citations1996Molly M. He, Jianzhong Sun +1 more
Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain XII and the periplasmic loop between putative helices XI and XII was replaced individually with Cys to demonstrate that transmemBRane domain XI is in an alpha-helical conformation.
BiochemistrySite-Directed Spin-Labeling of Transmembrane Domain VII and the 4B1 Antibody Epitope in the Lactose Permease of <i>Escherichia coli</i>
30 Citations1997John C. Voss, Wayne L. Hubbell +2 more
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.
Journal of Biological ChemistryCharacterization of purified, reconstituted site-directed cysteine mutants of the lactose permease of Escherichia coli
30 Citations1991Pierre R. van Iwaarden, Arnold J. M. Driessen +3 more
The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism and it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.
BiochemistryDynamics of Lactose Permease of Escherichia coli Determined by Site-Directed Chemical Labeling and Fluorescence Spectroscopy
29 Citations1995Jian Wu, Stathis Frillingos +1 more
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.
Protein ScienceThe role of helix VIII in the lactose permease of<i>Escherichia coli</i>: I. Cys‐scanning mutagenesis
29 Citations1997Stathis Frillingos, M. L. Ujwal +2 more
Results indicate that transmembrane domain VIII is in α‐helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism.
Journal of Biological ChemistryFunctional role of arginine 302 within the lactose permease of Escherichia coli.
29 Citations1992E.A. Matzke, L.J. Stephenson +1 more
Comparing the wild-type and mutant strains concerning their steady state pH gradient and their rates of H+ influx following oxygen pulses suggests that mutations at position 302 cause a sugar-dependent H+ leak.
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