Cysteine scanning mutagenesis of putative helix XI in the lactose permease of Escherichia coli

Biochemistry ◽  
1993 ◽  
Vol 32 (47) ◽  
pp. 12644-12650 ◽  
Author(s):  
Rhonda L. Dunten ◽  
Miklos Sahin-Toth ◽  
H. Ronald Kaback
Keyword(s):  
Escherichia Coli ◽  
Lactose Permease ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis

scholarly journals Cysteine scanning mutagenesis of the N-terminal 32 amino acid residues in the lactose permease of Escherichia coli

1994 ◽  
Vol 3 (2) ◽  
pp. 240-247 ◽  
Author(s):  
Miklós Sahin-Tóth ◽  
Bengt Persson ◽  
Jeremy Schwieger ◽  
Pejman Cohan ◽  
H.Ronald Kaback
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Lactose Permease ◽  
Amino Acid Residues ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis

Cysteine-Scanning Mutagenesis of Putative Helix VII in the Lactose Permease of Escherichia coli

Biochemistry ◽  
1994 ◽  
Vol 33 (26) ◽  
pp. 8074-8081 ◽  
Author(s):  
Stathis Frillingos ◽  
Miklos Sahin-Toth ◽  
Bengt Persson ◽  
H. Ronald Kaback
Keyword(s):  
Escherichia Coli ◽  
Lactose Permease ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis

scholarly journals Use of the transport specificity ratio and cysteine-scanning mutagenesis to detect multiple substrate specificity determinants in the consensus amphipathic region of the Escherichia coli GABA (γ-aminobutyric acid) transporter encoded by gabP

2003 ◽  
Vol 376 (3) ◽  
pp. 633-644 ◽  
Author(s):  
Steven C. KING ◽  
Lisa BROWN-ISTVAN
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Chemical Potential ◽  
Hydrophobic Surface ◽  
Structural Features ◽  
Aminobutyric Acid ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis ◽  
Specificity Determinants ◽  
Substrate Discrimination

The Escherichia coli GABA (γ-aminobutyric acid) permease, GabP, and other members of the APC (amine/polyamine/choline) transporter superfamily share a CAR (consensus amphipathic region) that probably contributes to solute translocation. If true, then the CAR should contain structural features that act as determinants of substrate specificity (kcat/Km). In order to address this question, we have developed a novel, expression-independent TSR (transport specificity ratio) analysis, and applied it to a series of 69 cysteine-scanning (single-cysteine) variants. The results indicate that GabP has multiple specificity determinants (i.e. residues at which an amino acid substitution substantially perturbs the TSR). Specificity determinants were found: (i) on a hydrophobic surface of the CAR (from Leu-267 to Ala-285), (ii) on a hydrophilic surface of the CAR (from Ser-299 to Arg-318), and (iii) in a cytoplasmic loop (His-233) between transmembrane segments 6 and 7. Overall, these observations show that (i) structural features within the CAR have a role in substrate discrimination (as might be anticipated for a transport conduit) and, interestingly, (ii) the substrate discrimination task is shared among specificity determinants that appear too widely dispersed across the GabP molecule to be in simultaneous contact with the substrates. We conclude that GabP exhibits behaviour consistent with a broadly applicable specificity delocalization principle, which is demonstrated to follow naturally from the classical notion that translocation occurs synchronously with conformational transitions that change the chemical potential of the bound ligand [Tanford (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2882–2884].

Putative identification of an amphipathic α-helical sequence in hemolysin of Escherichia coli (HlyA) involved in transmembrane pore formation

2008 ◽  
Vol 389 (9) ◽  
Author(s):  
Angela Valeva ◽  
Isabel Siegel ◽  
Mark Wylenzek ◽  
Trudy M. Wassenaar ◽  
Silvia Weis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Pore Formation ◽  
Cysteine Residue ◽  
Binding Properties ◽  
Cysteine Scanning ◽  
Helix Structure ◽  
Cysteine Scanning Mutagenesis ◽  
Α Helix ◽  
Polarity Sensitive

AbstractEscherichia colihemolysin is a pore-forming protein belonging to the RTX toxin family. Cysteine scanning mutagenesis was performed to characterize the putative pore-forming domain of the molecule. A single cysteine residue was introduced at 48 positions within the sequence spanning residues 170–400 and labeled with the polarity-sensitive dye badan. Spectrofluorimetric analyses indicated that several amino acids in this domain are inserted into the lipid bilayer during pore formation. An amphipathic α-helix spanning residues 272–298 was identified that may line the aqueous pore. The importance of this sequence was highlighted by the introduction of two prolines at positions 284 and 287. Disruption of the helix structure did not affect binding properties, but totally abolished the hemolytic activity of the molecule.

Cysteine-Scanning Mutagenesis of Helix II and Flanking Hydrophilic Domains in the Lactose Permease ofEscherichia coli

Biochemistry ◽  
1997 ◽  
Vol 36 (1) ◽  
pp. 269-273 ◽  
Author(s):  
Stathis Frillingos ◽  
Jianzhong Sun ◽  
Alberto Gonzalez ◽  
H. Ronald Kaback
Keyword(s):  
Ofescherichia Coli ◽  
Lactose Permease ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis
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