The role of helix VIII in the lactose permease of <i>Escherichia coli</i>: II. Site‐directed sulfhydryl modification

Frillingos, Stathis; Kaback, H. Ronald

doi:10.1002/pro.5560060221

Cited by 45 publications

(62 citation statements)

References 32 publications

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“…Therefore, taken as a whole, the observations imply that one face of helix VI11 plays an important role in the transport mechanism. More direct evidence for this conclusion is provided in the companion paper (Frillingos & Kaback, 1997).…”

Section: Discussionmentioning

confidence: 77%

“…7). In the companion paper (Frillingos & Kaback, 1997). further evidence for this argument is presented.…”

Section: Discussionmentioning

confidence: 88%

“…NEM is membrane-permeant and a number of single-Cys permease mutants located within transmembrane domains and disposed toward the inner surface of the membrane are inhibited readily (Sahin-T6th et al. 1992 (Frillingos & Kaback, 1996b: Frillingos & Kaback. 1997.…”

Section: Discussionmentioning

confidence: 99%

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The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis

Frillingos¹,

Ujwal²,

Sun³

et al. 1997

Protein Science

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Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VI11 and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to >70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262 4 Cys, Gly 268 + Cys, Asn 272 + Cys, Pro 280 + Cys, Asn 284 + Cys, Gly 287 + Cys, and Gly 288 + Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As a-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. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S , Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ. Keywords: active transport; bioenergetics; Cys modification; Cys replacementThe lactose permease of Escherichin coli, which catalyzes the coupled stoichiometric translocation of P-galactosides and H+ as a monomer, is an integral membrane protein with 12-transmembrane domains that are most probably in a-helical conformation (reviewed in . Site-directed mutagenesis of wild-type permease and Cys-scanning mutagenesis of a functional mutant devoid of Cys residues (C-less permease) have provided important insights into the structure and mechanism of the permease. Although as few as four amino acid residues [Glu 269 (helix VIII), Arg 302 (helix IX), His 322 (helix X), and Glu 325 (helix X)] appear to be critically involved in the symport mechanism, the activity of various active Cys-replacement mutants is altered by alkylation, and these mutants appear in clusters, suggesting that helical interfaces within the permease are important for turnover. Moreover, site-directed fluorescence spectroscopy (Jung et al

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Section: Discussionmentioning

confidence: 77%

“…7). In the companion paper (Frillingos & Kaback, 1997). further evidence for this argument is presented.…”

Section: Discussionmentioning

confidence: 88%

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The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis

Frillingos¹,

Ujwal²,

Sun³

et al. 1997

Protein Science

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“…Cells were washed twice in assay buffer (30 mM MES, pH 5.6, 2% glucose), and resuspended at 20 OD 600 /ml in the same buffer. Uptake of [2, H]-inosine (American Radiolabeled Chemicals, Inc., St. Louis, MO) was measured by the oil-stop method [25] …”

Section: Inosine Uptake Assays On Live Yeast Cellsmentioning

confidence: 99%

“…Several of these methods make use of strategically placed cysteine residues because of their unique chemistry among the natural amino acids. For example, cysteines engineered into two transmembrane helices that can be cross-linked with bifunctional thiol-reactive reagents of varying lengths provide information about the distance between the two helices in the folded structure of the protein [2]. Also, the steric and chemical environment of a cysteine residue engineered at a position of interest can be probed by the substituted cysteine accessibility method (SCAM), fluorescence resonance energy transfer (FRET), or site-directed spin labeling using a battery of thiol-reactive reagents of diverse sizes, chemical characteristics, and functionalities [3][4][5][6].…”

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confidence: 99%

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

Arendt

Yates

et al. 2007

Analytical Biochemistry

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We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site-saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2, in order to facilitate biochemical studies using thiolspecific modifying reagents. Of ten endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single-and double-mutant libraries were produced by combining a previously reported site-saturation mutagenesis scheme based on the Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in S. cerevisiae cells auxotrophic for purines, several highly functional non-cysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site-saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position. Keywordscysteine-less; substituted cysteine accessibility method; membrane protein; site-saturation mutagenesis; genetic selection; Quikchange mutagenesis; equilibrative nucleoside transporter A detailed understanding of how a protein functions must include structural information at some level of resolution. While x-ray crystal structures of thousands of soluble proteins have been determined to date, crystals of membrane proteins that diffract to high resolution are notoriously difficult to obtain [1]. In the absence of any high-resolution structural information for many membrane protein families, biochemical methods provide a valuable means to intuit structure. Several of these methods make use of strategically placed cysteine residues because of their unique chemistry among the natural amino acids. For example, cysteines engineered into two transmembrane helices that can be cross-linked with bifunctional thiol-reactive reagents of varying lengths provide information about the distance between the two helices in the folded structure of the protein [2]. Also, the steric and chemical environment of a cysteine residue engineered at a position of interest can be probed by the substituted cysteine Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [3][...

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Helix proximity and ligand-induced conformational changes in the lactose permease of Escherichia coli determined by site-directed chemical crosslinking

Kaback

1997

Journal of Molecular Biology

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The role of helix VIII in the lactose permease of Escherichia coli: II. Site‐directed sulfhydryl modification

Cited by 45 publications

References 32 publications

The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis

The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

Helix proximity and ligand-induced conformational changes in the lactose permease of Escherichia coli determined by site-directed chemical crosslinking

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