Purification of Adapted Reaction Centers

Details of the designs of adapted RCs are described in Supplementary Note 1. All adapted RCs were expressed in a strain of Rba. sphaeroides engineered to lack light-harvesting complexes^{54 (link),55 (link)}. Bacterial cells grown under dark/semiaerobic conditions and harvested by centrifugation were suspended in 20 mM tris(hydroxymethyl)aminomethane (Tris, pH 8.0), supplemented with protease inhibitor and DNAase, and lysed in a cell disruptor (Constant Systems) at 20,000 psi. Cell debris was removed by centrifugation at 18,000 rpm for 15 min at 4 °C. The supernatant was then incubated in the dark at 4 °C for 1 h with 1.5% (v/v) lauryldimethylamine N-oxide (LDAO) and 200 mM NaCl. After ultracentrifugation at 38,000 rpm for 30 min at 4 °C the supernatant was collected and initial purification carried out using a Ni-NTA (nitrilotriacetic acid) column (GE Healthcare). The protein eluate was concentrated and then further purified using a Superdex 200 gel filtration column (GE Healthcare) in 20 mM Tris (pH 8) containing 0.04% N-dodecyl-β-d-maltopyranoside (Tris/DDM buffer). Eluted fractions with a low 280 to 804 nm absorbance ratio (<1.5) were pooled, concentrated and stored at −80 °C.