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.

scholarly journals Cysteine Scanning Mutagenesis of α4, a Putative Pore-Lining Helix of the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

2008 ◽  
Vol 74 (9) ◽  
pp. 2565-2572 ◽  
Author(s):  
Frédéric Girard ◽  
Vincent Vachon ◽  
Gabrielle Préfontaine ◽  
Lucie Marceau ◽  
Yanhui Su ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Membrane Vesicles ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Insect Larvae ◽  
Insecticidal Toxin ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis ◽  
Pore Lining

ABSTRACT Helix α4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the α4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.

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].

scholarly journals Mechanism of ATP-sensitive K Channel Inhibition by Sulfhydryl Modification

1998 ◽  
Vol 112 (3) ◽  
pp. 325-332 ◽  
Author(s):  
Stefan Trapp ◽  
Stephen J. Tucker ◽  
Frances M. Ashcroft
Keyword(s):  
Katp Channel ◽  
Membrane Surface ◽  
Katp Channels ◽  
Cysteine Residue ◽  
K Channel ◽  
Atp Binding Site ◽  
Channel Inhibition ◽  
Cysteine Scanning ◽  
Sulfhydryl Modification ◽  
Cysteine Scanning Mutagenesis

ATP-sensitive potassium (KATP) channels are reversibly inhibited by intracellular ATP. Agents that interact with sulfhydryl moieties produce an irreversible inhibition of KATP channel activity when applied to the intracellular membrane surface. ATP appears to protect against this effect, suggesting that the cysteine residue with which thiol reagents interact may either lie within the ATP-binding site or be inaccessible when the channel is closed. We have examined the interaction of the membrane-impermeant thiol-reactive agent p-chloromercuriphenylsulphonate (pCMPS) with the cloned β cell KATP channel. This channel comprises the pore-forming Kir6.2 and regulatory SUR1 subunits. We show that the cysteine residue involved in channel inhibition by pCMPS resides on the Kir6.2 subunit and is located at position 42, which lies within the NH2 terminus of the protein. Although ATP protects against the effects of pCMPS, the ATP sensitivity of the KATP channel was unchanged by mutation of C42 to either valine (V) or alanine (A), suggesting that ATP does not interact directly with this residue. These results are consistent with the idea that C42 is inaccessible to the intracellular solution, and thereby protected from interaction with pCMPS when the channel is closed by ATP. We also observed that the C42A mutation does not affect the ability of SUR1 to endow Kir6.2 with diazoxide sensitivity, and reduces, but does not prevent, the effects of MgADP and tolbutamide, which are mediated via SUR1. The Kir6.2-C42A (or V) mutant channel may provide a suitable background for cysteine-scanning mutagenesis studies.

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 Cysteine-Scanning Mutagenesis Supports the Importance of Clostridium perfringens Enterotoxin Amino Acids 80 to 106 for Membrane Insertion and Pore Formation

2012 ◽  
Vol 80 (12) ◽  
pp. 4078-4088 ◽  
Author(s):  
Jianwu Chen ◽  
James R. Theoret ◽  
Archana Shrestha ◽  
James G. Smedley ◽  
Bruce A. McClane
Keyword(s):  
Amino Acids ◽  
Clostridium Perfringens ◽  
Pore Formation ◽  
Gastrointestinal Symptoms ◽  
Amino Acid Residues ◽  
Content Type ◽  
Food Borne ◽  
Food Borne Illness ◽  
Cysteine Scanning Mutagenesis

ABSTRACTClostridium perfringensenterotoxin (CPE) causes the gastrointestinal symptoms of the second most common bacterial food-borne illness. Previous studies suggested that a region named TM1, which has amphipathic characteristics and spans from amino acids 81 to 106 of the native CPE protein, forms a β-hairpin involved in β-barrel pore formation. To further explore the potential role of TM1 in pore formation, the single Cys naturally present in CPE at residue 186 was first altered to alanine by mutagenesis; the resultant rCPE variant, named C186A, was shown to retain cytotoxic properties. Cys-scanning mutagenesis was then performed in which individual Cys mutations were introduced into each TM1 residue of the C186A variant. When those Cys variants were characterized, three variants were identified that exhibit reduced cytotoxicity despite possessing binding and oligomerization abilities similar to those of the C186A variant from which they were derived. Pronase challenge experiments suggested that the reduced cytotoxicity of those two Cys variants, i.e., the F91C and F95C variants, which model to the tip of the β-hairpin, was attributable to a lessened ability of these variants to insert into membranes after oligomerization. In contrast, another Cys variant, i.e., the G103C variant, with impaired cytotoxicity apparently inserted into membranes after oligomerization but could not form a pore with a fully functional channel. Collectively, these results support the TM1 region forming a β-hairpin as an important step in CPE insertion and pore formation. Furthermore, this work identifies the first amino acid residues specifically involved in those two steps in CPE action.

Investigation of the interaction of batatasin derivatives with human serum albumin using voltammetric and spectroscopic methods

RSC Advances ◽  
2016 ◽  
Vol 6 (43) ◽  
pp. 36281-36292 ◽  
Author(s):  
Jinhua Zhu ◽  
Weiping Hu ◽  
Dandan Wu ◽  
Lanlan Chen ◽  
Xiuhua Liu
Keyword(s):  
Molecular Docking ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Spectroscopic Methods ◽  
Binding Properties ◽  
Helix Structure ◽  
Α Helix

The binding properties of batatasin derivatives with HSA were estimated by voltammetric, spectroscopic, and molecular docking methods. There were non-electroactive complexes formed between them. And the α-helix structure in HSA was reduced.

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