Strategy for the Thermostabilization of an Agonist-Bound GPCR Coupled to a G Protein

• Published in: Methods in enzymology Vol. 594; p. 243
• Main Authors: Strege, Annette, Carpenter, Byron, Edwards, Patricia C, Tate, Christopher G
• Format: Journal Article
• Language: English
• Published: United States 2017
• Subjects: Amphibian Proteins - chemistry; Biochemistry - methods; GTP-Binding Protein alpha Subunits, Gs - chemistry; GTP-Binding Protein alpha Subunits, Gs - metabolism; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - metabolism; Heterotrimeric GTP-Binding Proteins - agonists; Heterotrimeric GTP-Binding Proteins - chemistry; Heterotrimeric GTP-Binding Proteins - metabolism; Humans; Iodine Radioisotopes - chemistry; Peptide Hormones - chemistry; Protein Stability; Receptors, Corticotropin-Releasing Hormone - agonists; Receptors, Corticotropin-Releasing Hormone - chemistry; Receptors, Corticotropin-Releasing Hormone - metabolism; G protein-coupled receptor; Stability; Structure; Mini-G protein
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2017.05.014

Structure determination of G protein-coupled receptors (GPCRs) in the inactive state bound to high-affinity antagonists has been very successful through the implementation of a number of protein engineering and crystallization strategies. However, the structure determination of GPCRs in their fully active state coupled to a G protein is still very challenging. Recently, mini-G proteins were developed, which recapitulate the coupling of a full heterotrimeric G protein to a GPCR despite being less than one-third of the size. This allowed the structure determination of the agonist-bound adenosine A receptor (A R) coupled to mini-G . Although this is extremely encouraging, A R is very stable compared with many other GPCRs, particularly when an agonist is bound. In contrast, the agonist-bound conformation of the human corticotropin-releasing factor receptor is considerably less stable, impeding the formation of good quality crystals for structure determination. We have therefore developed a novel strategy for the thermostabilization of a GPCR-mini-G protein complex. In this chapter, we will describe the theoretical and practical principles of the thermostability assay for stabilizing this complex, discuss its strengths and weaknesses, and show some typical results from the thermostabilization process.