Protocol for measuring β-arrestin1-phosphorylated peptide interaction and analyzing conformational dynamics of β-arrestin1

Arrestins associate with phosphorylated G protein-coupled receptors and undergo conformational changes. Here, we present a protocol for measuring β-arrestin1-phosphorylated peptide interaction and analyzing conformational dynamics of β-arrestin1. We describe steps for constructing expression plasmids, expressing and purifying β-arrestin1, and performing hydrogen/deuterium exchange mass spectrometry analysis of V2Rpp-β-arrestin1 complex. We then detail procedures for measuring binding affinity using microscale thermophoresis. This protocol has potential application in analyzing conformational dynamics and measuring binding affinity of arrestins. For complete details on the use and execution of this protocol, please refer to Kim et al.¹