G protein-coupled receptors (GPCRs) perform vital signaling functions and are involved in various
diseases, making GPCRs major drug targets. GPCRs have seven α-helical transmembrane domains connected
by three extracellular loops (ECLs) and three intracellular loops (ICLs). Among the three ICLs, ICL3 has
been reported to have a critical function in interacting with downstream signaling molecules. Despite its important
role in GPCR signaling, the structure of ICL3 has not been fully defined. In the present study, we
used muscarinic acetylcholine receptor type 1 (M1) as a model system to analyze the structure of ICL3. Optimized
purification conditions for M1_ICL3 comprised His-tag affinity purification and solubilization with
n-dodecyl-b-D-maltopyranoside. Purified M1_ICL3 was analyzed using circular dichroism and hydrogen/deuterium exchange
mass spectrometry; the results of these analyses suggested that M1_ICL3 is disordered and flexible.
Keywords: G protein-coupled receptor, hydrogen/deuterium exchange mass spectrometry, intracellular loop 3, muscarinic acetylcholine
receptor, protein conformation, protein purification.
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