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1.
Figure 2

Figure 2. From: Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p.

Schematic view of the procedures for mutagenesis and screening. The abscissas and ordinates of the dot plots represent the fluorescence detected in the FL1 and FL2 channels of a flow cytometer. Each dot represents a cell.

Elizabeth Mathew, et al. J Mol Biol. ;409(4):513-528.
2.
Figure 3

Figure 3. From: Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p.

[Lys7(NBD),Nle12]α-factor binding curves for selected Ste2p mutants from each of the four libraries. The closed symbols and the unbroken lines represent information obtained from the flow cytometer channel FL1. The open symbols and broken lines represent FL2.

Elizabeth Mathew, et al. J Mol Biol. ;409(4):513-528.
3.
Figure 1

Figure 1. From: Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p.

Predicted transmembrane topology of C-terminally truncated Ste2p. The black circles indicate residues that, when mutated, alter the FL1/FL2 ratio of fluorescence emission of [Lys7(NBD),Nle12]α-factor bound to C-terminally truncated Ste2p receptor. The gray circles indicate residues that, when mutated, alter the FL1/FL2 ratio of fluorescence emission of [dTyr3,Lys7(NBD),Nle12]α-factor bound to Ste2p receptor. The cross-hatched residue (F55) alters fluorescence ratio of both types of ligand. The arrows indicate the start- and end- points of the regions targeted for mutagenesis in the four screened libraries.

Elizabeth Mathew, et al. J Mol Biol. ;409(4):513-528.
4.
Figure 4

Figure 4. From: Differential Interactions of Fluorescent Agonists and Antagonists with the Yeast G Protein Coupled Receptor Ste2p.

Model for the binding and activation of normal and mutant α-factor receptors by different ligands. Ligand-interacting surfaces on the receptor are indicated by cross-hatching. Site I interacts with the C-termini of ligands and is likely to consist of the extracellular end of the first transmembrane segment. Site 2 interacts with the N-termini of ligands and most likely consists of the extracellular end of the sixth transmembrane segment and the third extracellular loop of the receptor (see 13;14;15;16;17;18;32). The conformational equilibrium between inactive and activated states of the receptor is schematically represented as a change in the distance between Site 1 and Site 2. a) In the absence of ligand, the receptor favors the unactivated state with a large separation between ligand contact sites. b) Binding of agonist to normal receptors favors the activated state. c) Mutations affecting the emission spectrum of bound agonist change the environment of the fluorophore (red-shift) without altering receptor ligand binding or, generally, activation. d) Binding of ligands that act as antagonists toward normal receptors (labeled as “antagonist”) does not alter the receptor’s conformational equilibrium. Activation-specific interactions with the N-terminal of the ligand are blocked by the dTyr3 substitution (orange circle), leaving the NBD group attached at Lys7 exposed to solvent while the C-terminal region of the antagonist (which is identical to the corresponding region of agonist) maintains a high affinity interaction with receptor. e) Mutations that restore receptor binding to the altered N-terminal region of antagonist allow signaling responses to “antagonist” ligands. The altered binding mode to the mutant receptors allows the NBD group to be protected from solvent. f) Mutations that constitutively activate the receptor by favoring the activated conformation, enhancing the interaction of Site 2 of the receptor with antagonists that exhibit weak partial agonist activity. The altered binding mode allows the NBD group to be protected from solvent.

Elizabeth Mathew, et al. J Mol Biol. ;409(4):513-528.

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