Stabilization and structure determination of integral membrane proteins by termini restraining

Integral membrane proteins isolated from cellular environment often lose activity and native conformation required for functional analyses and structural studies. Even in their native state, they lack sufficient surfaces to form crystal contacts. Furthermore, most of them are too small for cryogenic electron microscopy detection and too big for solution NMR. To overcome these difficulties, we recently developed a strategy to stabilize the folded state of membrane proteins by restraining their two termini with a self-assembling protein coupler. The termini-restrained membrane proteins from distinct functional families retain their activities and show increased stability and yield. This strategy enables their structure determination at near-atomic resolution by facilitating the entire pipeline from crystallization, crystal identification, diffraction enhancement and phase determination, to electron density improvement. Furthermore, stabilization of membrane proteins enables their biochemical and biophysical characterization. Here we present the protocol of membrane protein engineering (2 weeks), quality assessment (1–2 weeks), protein production (1–6 weeks), crystallization (1–2 weeks), diffraction improvement (1–3 months) and crystallographic data analysis (1 week). This protocol is intended not only for structural biologists, but also for biochemists, biophysicists and pharmaceutical scientists whose research focuses on membrane proteins.