Site-directed spin labeling

Cysteine was substituted for selected residues in the recombinant HAMP domain with QuikChange (Agilent Genomics, Santa Clara, CA) mutagenesis or overlap extension. The purified proteins were reacted with cysteine-specific nitroxide S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate spin-label (MTSL; Toronto Research Chemicals, North York, Ontario, Canada) on a Ni-NTA column for 4 h at room temperature, and subsequently at 4°C for 8 h. The proteins were eluted with 20 mM Tris (pH 8.0), 250 mM imidazole (pH 8.0), 500 mM NaCl, and 10% (v/v) glycerol. The samples were subjected to size-exclusion chromatography (Superdex 75 26–60) for further purification and removal of unreacted MTSL.