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 Induction of substrate specificity shifts by placement of alanine insertions within the consensus amphipathic region of the Escherichia coli GABA (γ-aminobutyric acid) transporter encoded by gabP

2003 ◽  
Vol 376 (3) ◽  
pp. 645-653 ◽  
Author(s):  
Steven C. KING ◽  
Liaoyuan A. HU ◽  
Amy PUGH
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Hydrophobic Surface ◽  
Aminobutyric Acid ◽  
Insertion Mutagenesis ◽  
Nipecotic Acid ◽  
E Coli ◽  
Gaba Accumulation ◽  
Thermodynamic Basis ◽  
Specificity Determinants

The Escherichia coli GABA (γ-aminobutyric acid) permease GabP is a prototypical APC (amine/polyamine/choline) super-family transporter that has a CAR (consensus amphipathic region) containing multiple specificity determinants, ostensibly organized on two helical surfaces, one hydrophobic [SHS (sensitive hydrophobic surface)] and the other hydrophilic [SPS (sensitive polar surface)]. To gauge the functional effects of placing alanine insertions at close intervals across the entire GabP CAR, 64 insertion variants were constructed. Insertions, particularly those in the SHS and the SPS, were highly detrimental to steady-state [3H]GABA accumulation. TSR (transport specificity ratio) analysis, employing [3H]nipecotic acid and [14C]GABA, showed that certain alanine insertions were associated with a specificity shift (i.e. a change in kcat/Km). An insertion (INS Ala-269) located N-terminal to the SHS increased specificity for [3H]nipecotic acid relative to [14C]GABA, whereas an insertion (INS Ala-321) located C-terminal to the SPS had the opposite effect. Overall, the results are consistent with a working hypothesis that the GabP CAR contains extensive functional surfaces that may be manipulated by insertion mutagenesis to alter the specificity (kcat/Km) phenotype. The thermodynamic basis of TSR analysis provides generality, suggesting that amino acid insertions could affect specificity in many other transporters, particularly those such as the E. coli phenylalanine permease PheP [Pi, Chow and Pittard (2002) J. Bacteriol. 184, 5842–5847] that have a functionally significant CAR-like domain.

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 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 Organization of ATP-gated P2X1 receptor intracellular termini in apo and desensitized states

2019 ◽  
Vol 151 (2) ◽  
pp. 146-155 ◽  
Author(s):  
Alistair G. Fryatt ◽  
Sudad Dayl ◽  
Anastasios Stavrou ◽  
Ralf Schmid ◽  
Richard J. Evans
Keyword(s):  
Time Course ◽  
Structural Features ◽  
Hek293 Cells ◽  
P2x1 Receptor ◽  
C Terminus ◽  
Close Proximity ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis ◽  
Intracellular Domains ◽  
Control Construct

The human P2X1 receptor (hP2X1R) is a trimeric ligand-gated ion channel opened by extracellular ATP. The intracellular amino and carboxyl termini play significant roles in determining the time-course and regulation of channel gating—for example, the C terminus regulates recovery from the desensitized state following agonist washout. This suggests that the intracellular regions of the channel have distinct structural features. Studies on the hP2X3R have shown that the intracellular regions associate to form a cytoplasmic cap in the open state of the channel. However, intracellular features could not be resolved in the agonist-free apo and ATP-bound desensitized structures. Here we investigate the organization of the intracellular regions of hP2X1R in the apo and ATP-bound desensitized states following expression in HEK293 cells. We couple cysteine scanning mutagenesis of residues R25-G30 and H355-R360 with the use of bi-functional cysteine reactive cross-linking compounds of different lengths (MTS-2-MTS, BMB, and BM(PEG)2), which we use as molecular calipers. If two cysteine residues come into close proximity, we predict they will be cross-linked and result in ∼66% of the receptor subunits running on a Western blot as dimers. In the control construct (C349A) that removed the free cysteine C349, and some cysteine-containing mutants, cross-linker treatment does not result in dimerization. However, we detect efficient dimerization for R25C, G30C, P358C, K359C, and R360C. This selective pattern indicates that there is structural organization to these regions in the apo and desensitized states in a native membrane environment. The existence of such precap (apo) and postcap (desensitized) organization of the intracellular domains would facilitate efficient gating of the channel.

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
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