Construct σ1long, comprising the three most C-terminal β-spirals of T1L σ1 and the entire head domain (amino acids 261 to 470), was purified as described previously (22 (link)) and used in STD-NMR experiments. Mutations were generated using the GeneArt site-directed mutagenesis PLUS system (Invitrogen) along with AccuPrime Pfx DNA polymerase (Invitrogen) and One Shot MAX Efficiency DH5α-T1R competent cells (Invitrogen).