Isolated α_M and α_D I-domains directly bind to CEP in surface plasmon resonance assays. (A) SDS-PAGE of generated I-domains. The I-domains α_L active (G¹²⁷-Y³⁰⁷), α_D nonactive (P¹²⁸-S³²³), α_D active (D¹²²-K³¹⁴), α_M nonactive (Q¹¹⁹-E³³³), and α_M active (E¹²³-K³¹⁵) were isolated from soluble fractions of Escherichia coli lysates, purified using affinity chromatography, and their purity assessed by SDS-PAGE on 4% to 20% gradient gel under reducing conditions followed by staining with Coomassie Blue. (B-G) Representative profiles of the SPR responses for α_M (B) and α_D (C) binding to the immobilized CEP-BSA and EP-BSA. Binding of α_M I-domain (D) and α_D I-domain (E) in active conformation (concentrations ranging from 16 to 500 nM) to CEP-BSA coupled to the CM5 chip. (F) α_M I-domain in nonactive conformation has significantly reduced binding to CEP-BSA. (G) α_D I-domain in nonactive conformation and α_L I-domain in active conformation demonstrate very low binding to CEP-BSA. PAGE, polyacrylamide gel electrophoresis; SPR, surface plasmon resonance.