Sexual conjugation in yeast: A paradigm to study G‐protein‐coupled receptor domain structure

Abstract

The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells. The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors. The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure. Using chemical procedures we have synthesized peptides corresponding to the seven transmembrane domains of Ste2p and studied their structures in membrane mimetic environments. Extension of these studies requires preparation of longer fragments of Ste2p. This article discusses strategies used in our laboratory to prepare peptides containing multiple domains of Ste2p. Data are presented on the use of chemical synthesis, biosynthesis, and native chemical ligation. Using biosynthetic approaches fusion proteins have been expressed that contain single receptor domains, two transmembrane domains connected by the contiguous loop, and the tail connected to the seventh transmembrane domain. Tens of milligrams of fusion protein were obtained per liter, and multimilligram quantities of the isotopically labeled target peptides were isolated using such biosynthetic approaches. Initial circular dichroism results on a chemically synthesized 64-residue peptide containing a portion of the cytosolic tail and the complete seventh transmembrane domain showed that the tail portion and the hydrophobic core of this peptide maintained individual conformational preferences. Moreover, this peptide could be studied at near millimolar concentrations in the presence of micelles and did not aggregate under these conditions. Thus, these constructs can be investigated using high-resolution nuclear magnetic resonance techniques, and the cytosolic tail of Ste2p can be used as a hydrophilic template to improve solubility of transmembrane peptides for structural analysis.

Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p

“…Both receptors couple to the same heterotrimeric Gprotein composed of Gα (Gpa1p), Gβ (Ste4p) and Gγ (Ste18p), and stimulation results in the release of the Gβγ dimer, which then interacts with effectors to bring about conjugation. α-Factor receptor (Ste2p) is one of the prototypical GPCRs that has been widely used as a model system for studying structure-function relationships of ligand-GPCR interaction, the mechanism of G-protein activation upon ligand binding and the regulation of downstream signaling processes via heterotrimeric G-protein [6].…”

At Last: Erythropoietin as a Single Glycoform

“…19), the use of trifluoroethanol (TFE) as co-solvent in the final step was critical for the success of the final transformation. [28] …”

A transmembrane helix‐bundle from G‐protein coupled receptor CB2: Biosynthesis, purification, and NMR characterization

“…More recently, two doublehelix fragments from GPCRs were expressed and structural investigations were carried out by NMR and other biophysical methods. 20,25 Therefore, the preparation and study of transmembrane helix-bundles of GPCRs not only can facilitate determination of the complete GPCR structure, but also can provide new information concerning the physical properties, biological activities, and therapeutic potentials of these unique biopolymers.…”

“…20,26 Transmembrane domains are expressed as fusion proteins with a leading tag TrpLED1413 (TrpLE) 27 that promotes the formation of inclusion bodies. Using this approach we have overexpressed, isolated, purified, and characterized two transmembrane segments, CB2(65-101) 15 and CB2(180-233), 10 which correspond to TM2 and TM5-i3 regions of the CB2 receptor.…”

“…[10][11][12][13][14][15] This ''from building blocks to assembly in 3D'' strategy to obtain GPCR NMR structures was proposed and utilized by several groups, including ours, to study GPCRs including rhodopsin, [16][17][18] the -factor receptor, [19][20][21] and the cannabinoid receptor CB2. [10][11][12][13][14][15] NMR studies of rhodopsin demonstrated that the peptide segments of local domains adopted secondary structures similar to those observed by X-ray crystallography 17,[22][23][24] ; rhodopsin is the only GPCR for which a crystal structure is available.…”